Pichia methanolica secretory signal

ABSTRACT

Novel  Pichia methanolica  secretory signal polypeptides, polynucleotides encoding the polypeptides, and related compositions and methods of using are disclosed. Methods of producing large amounts of recombinant proteins by employing DNA constructs having a polypeptide of interest preceded by a novel  Pichia methanolica  secretory signal sequence.

The present application is a divisional of U.S. patent application Ser.No. 10/903,350, filed Jul. 30, 2004, which claims the benefit of U.S.Patent Application Ser. Nos. 60/491,093, filed Jul. 30, 2003, and60/501,134, filed Sep. 8, 2003, all of which are herein incorporated byreference.

BACKGROUND OF THE INVENTION

Methylotrophic yeasts are those yeasts that are able to utilize methanolas a sole source of carbon and energy. Species of yeasts that have thebiochemical pathways necessary for methanol utilization are classifiedin four genera, Hansenula, Pichia, Candida, and Torulopsis. These generaare somewhat artificial, having been based on cell morphology and growthcharacteristics, and do not reflect close genetic relationships(Billon-Grand, Mycotaxon 35:201-204, 1989; Kurtzman, Mycologia 84:72-76,1992). Furthermore, not all species within these genera are capable ofutilizing methanol as a source of carbon and energy. As a consequence ofthis classification, there are great differences in physiology andmetabolism between individual species of a genus.

Methylotrophic yeasts are attractive candidates for use in recombinantprotein production systems for several reasons. First, somemethylotrophic yeasts have been shown to grow rapidly to high biomass onminimal defined media. Second, recombinant expression cassettes aregenomically integrated and therefore mitotically stable. Third, theseyeasts are capable of secreting large amounts of recombinant proteins.See, for example, Faber et al., Yeast 11:1331, 1995; Romanos et al.,Yeast 8:423, 1992; Cregg et al., Bio/Technology 11:905, 1993; U.S. Pat.No. 4,855,242; U.S. Pat. No. 4,857,467; U.S. Pat. No. 4,879,231; andU.S. Pat. No. 4,929,555; and Raymond, U.S. Pat. Nos. 5,716,808,5,736,383, 5,854,039, and 5,888,768.

In the commercial production of proteins via recombinant DNAtechnologies, it is often advantageous for the desired protein ofinterest to be secreted into the growth medium. Secretion of proteinsfrom cells is generally accomplished by the presence of a short stretchof hydrophobic amino acids constituting the amino-terminal end of thetranslational product. This hydrophobic stretch is call the “secretorysignal sequence,” and it is possible to use signal sequences to effectthe secretion of heterologous proteins. This is generally accomplishedby the construction of an DNA construct comprising a DNA sequenceencoding a secretory signal sequence, into which a gene encoding thedesired heterologous protein is inserted. When such a plasmid istransformed into a host cell, the host cell will express and secrete thedesired protein into the growth medium.

At present, the only mode of achieving secretion of a heterologousprotein product in Pichia methanolica is by way of a foreign secretorysignal peptide. Because foreign gene's are not native to Pichiamethanolica, the levels of heterologous protein expression are likelysuboptimal as compared to a DNA construct incorporating a secretorysignal sequence native to Pichia methanolica.

Thus, there remains a need in the art to identify a secretory signalsequence native to Pichia methanolica to enable the use ofmethylotrophic yeasts for production of polypeptides of economicimportance, including industrial enzymes and pharmaceutical proteins.The present invention provides such materials and methods as well asother, related advantages.

DETAILED DESCRIPTION OF THE INVENTION

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

The term “allelic variant” is used herein to denote an alternative formof a gene. Allelic variation is known to exist in populations and arisesthrough mutation.

A “DNA construct” is a DNA molecule, either single- or double-stranded,that has been modified through human intervention to contain segments ofDNA combined and juxtaposed in an arrangement not existing in nature.

A “DNA segment” is a portion of a larger DNA molecule having specifiedattributes. For example, a DNA segment encoding a specified polypeptideis a portion of a longer DNA molecule, such as a plasmid or plasmidfragment, that, when read from the 5′ to the 3′ direction, encodes thesequence of amino acids of the specified polypeptide.

The term “functionally deficient” denotes the expression in a cell ofless than 10% of an activity as compared to the level of that activityin a wild-type counterpart. It is preferred that the expression level beless than 1% of the activity in the wild-type counterpart, morepreferably less than 0.01% as determined by appropriate assays. It ismost preferred that the activity be essentially undetectable (i.e., notsignificantly above background). Functional deficiencies in genes can begenerated by mutations in either coding or non-coding regions.

The term “gene” is used herein to denote a DNA segment encoding apolypeptide. Where the context allows, the term includes genomic DNA(with or without intervening sequences), cDNA, and synthetic DNA. Genesmay include non-coding sequences, including promoter elements.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones.

“Operably linked”, when referring to DNA segments, indicates that thesegments are arranged so that they function in concert for theirintended purposes, e.g., transcription initiates in the promoter andproceeds through the coding segment to the terminator.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theseterms are applied to double-stranded molecules they are used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes. Sequences within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, and transcription factor binding sites.See, in general, Watson et al., eds., Molecular Biology of the Gene, 4thed., The Benjamin/Cummings Publishing Company, Inc., Menlo Park, Calif.,1987.

A “pro sequence” is a DNA sequence that commonly occurs immediately 5′to the mature coding sequence of a gene encoding a secretory protein.The pro sequence encodes a pro peptide that serves as a cis-actingchaperone as the protein moves through the secretory pathway.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are commonly defined interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway. A secretory peptide and a pro peptide may becollectively referred to as a pre-pro peptide.

As used herein, a “therapeutic agent” is a molecule or atom which isconjugated to an antibody moiety to produce a conjugate which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes.

A “detectable label” is a molecule or atom which can be conjugated to anantibody moiety to produce a molecule useful for diagnosis. Examples ofdetectable labels include chelators, photoactive agents, radioisotopes,fluorescent agents, paramagnetic ions, or other marker moieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). Nucleic acid molecules encoding affinity tagsare available from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

All references cited herein are incorporated by reference in theirentirety.

At present, the only mode of achieving secretion of a heterologousprotein product in Pichia methanolica is by way of a foreign secretorysignal peptide. Because foreign gene's are not native to Pichiamethanolica, the levels of heterologous protein expression are likelysuboptimal as compared to a DNA construct incorporating a secretorysignal sequence native to Pichia methanolica. Without being limited to atheory, a native Pichia methanolica secretory signal peptide wouldincrease heterologous protein production by more effectively directingtransport of the heterologous protein to its target membrane, and bybeing cleaved more efficiently by Pichia methanolica peptidase on themembrane when the heterologous protein passes through it.

The present invention provides isolated DNA molecules comprising aPichia methanolica secretory signal sequence, designatedexo-1,3-β-glucanase gene and hereinafter referred to as “β-glucanase,”is shown in SEQ ID NO:1, the encoded polypeptide is shown in SEQ IDNO:2, and the degenerate DNA molecule encoding the polypeptide of SEQ IDNO:2 is shown in SEQ ID NO:3. Those skilled in the art will recognizethat SEQ ID NO:1 represents a single allele of the P. methanolicaβ-glucanase gene and that other functional alleles (allelic variants)are likely to exist, and that allelic variation may include nucleotidechanges. The β-glucanase DNA sequence may be included in a DNAconstruct. For example, a DNA construct can include the followingoperably linked elements, which include a Pichia methanolica promotersequence, β-glucanase DNA sequence, heterologous DNA sequence, and aPichia methanolica terminator.

An E. coli DH10B cell culture containing an expression vector encodingPichia methanolica secretory signal sequence β-glucanase was depositedwith the American Type Culture Collection (10801 University Boulevard,Manassas, Va. 20110-2209) on Aug. 1, 2003, and assigned Patent DepositDesignation No. PTA-5369. This deposit will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure. Thedeposit was made merely as a convenience for those of skill in the artand is not an admission that a deposit is required under 35 U.S.C. §112.

The present invention provides polynucleotide molecules, including DNAand RNA molecules, which encode the β-glucanase polypeptides disclosedherein. Those skilled in the art will readily recognize that, in view ofthe degeneracy of the genetic code, considerable sequence variation ispossible among these polynucleotide molecules. SEQ ID NO:3 is adegenerate DNA sequence that encompasses all DNAs that encode theβ-glucanase polypeptide, and fragments thereof, of SEQ ID NO:2. Thoseskilled in the art will recognize that the degenerate sequence of SEQ IDNO:3 also provides all RNA sequences encoding SEQ ID NO:2 bysubstituting U for T. Thus, β-glucanase polypeptide-encodingpolynucleotides comprising nucleotide 1 to nucleotide 84 of SEQ ID NO:3and their RNA equivalents are contemplated by the present invention.Table 1 sets forth the one-letter codes used within SEQ ID NO:3 todenote degenerate nucleotide positions. “Resolutions” are thenucleotides denoted by a code letter. “Complement” indicates the codefor the complementary nucleotide(s). For example, the code Y denoteseither C or T, and its complement R denotes A or G, with A beingcomplementary to T, and G being complementary to C. TABLE 1 NucleotideResolution Complement Resolution A A T T C C G G G G C C T T A A R A|G YC|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T W A|T H A|C|TD A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T NA|C|G|T

The degenerate codons used in SEQ ID NO:3, encompassing all possiblecodons for a given amino acid, are set forth in Table 2. TABLE 2 OneAmino Letter Degenerate Acid Code Codons Codon Cys C TGC,TGT TGY Ser SAGC, AGT, TCA, TCC, TCG, WSN TCT Thr T ACA, ACC, ACG, ACT ACN Pro PCCA,CCC,CCG,CCT CCN Ala A GCA, GCC, GCG, GCT GCN Gly G GGA,GGC,GGG,GGTGGN Asn N AAC, AAT AAY Asp D GAC, GAT GAY Glu E GAA, GAG GAR Gln Q CAA,CAG CAR His H CAC, CAT CAY Arg R AGA, AGG, CGA, CGC, CGG, MGN CGT Lys KAAA,AAG AAR Met M ATG ATG Ile I ATA, ATC, ATT ATH Leu L CTA, CTC, CTG,CTT, TTA, YTN TTG Val V GTA, GTC, GTG, GTT GTN Phe F TTC, TTT TTY Tyr YTAC, TAT TAY Trp W TGG TGG Ter . TAA, TAG, TGA TRR Asn|Asp B RAY Glu|GlnZ SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO:2. Variant sequences can be readily tested forfunctionality as described herein.

A full-length clone encoding β-glucanase can be obtained by conventionalcloning procedures. Complementary DNA (cDNA) clones are preferred,although for some applications (e.g., expression in transgenic animals)it may be preferable to use a genomic clone, or to modify a cDNA cloneto include at least one genomic intron. Methods for preparing cDNA andgenomic clones are well known and within the level of ordinary skill inthe art, and include the use of the sequence disclosed herein, or partsthereof, for probing or priming a library. Expression libraries can beprobed with antibodies to glucanse fragments, or other specific bindingpartners.

The present invention provides an isolated DNA molecule comprising anucleotide sequence of SEQ ID NO:1 or complement thereof. Those skilledin the art will recognize that the sequence disclosed in SEQ ID NO:1represents a single allele of human β-glucanase and that allelicvariation and alternative splicing are expected to occur. Allelicvariants of this sequence can be cloned by probing cDNA or genomiclibraries from different individuals according to standard procedures.Allelic variants of the DNA sequence shown in SEQ ID NO:1, includingthose containing silent mutations and those in which mutations result inamino acid sequence changes, are within the scope of the presentinvention, as are proteins which are allelic variants of SEQ ID NO:2.cDNAs generated from alternatively spliced mRNAs, which retain theproperties of the β-glucanase polypeptide, are included within the scopeof the present invention, as are polypeptides encoded by such cDNAs andmRNAs. Allelic variants and splice variants of these sequences can becloned by probing cDNA or genomic libraries from different individualsor tissues according to standard procedures known in the art.

The present invention also provides DNA molecules encoding apolypeptide, wherein the encoded polypeptide comprises an amino acidsequence having at least 95 percent sequence identity to SEQ ID NO:2,and wherein the encoded polypeptide is a secretory signal sequence ofPichia methanolica. The polypeptide may comprise, consist essentiallyof, or consist of SEQ ID NO:2.

The present invention also provides an isolated polypeptide comprisingan amino acid sequence having at least 95 percent sequence identity withSEQ ID NO:2, wherein the polypeptide is a secretory signal sequence ofPichia methanolica. The polypeptide may comprise, consist essentiallyof, or consist of SEQ ID NO:2.

The present invention also provides isolated β-glucanase polypeptidesthat have a substantially similar sequence identity to the polypeptidesof SEQ ID NO:2, or their orthologs. The term “substantially similarsequence identity” is used herein to denote polypeptides comprising atleast 70%, at least 80%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or greater than 99% sequenceidentity to the sequences shown in SEQ ID NO:2, or their orthologs. Thepresent invention also includes polypeptides that comprise an amino acidsequence having at least 70%, at least 80%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, or greater than99% sequence identity to the sequence of amino acid residues 1 to 28 ofSEQ ID NO:2. The present invention further includes DNA molecules thatencode such polypeptides. Methods for determining percent identity aredescribed below.

The present invention also provides a fusion protein comprising a firstportion and a second portion joined by a peptide bond, wherein the firstportion comprises an amino acid sequence of SEQ ID NO:2, and the secondportion comprises another polypeptide. The second portion may be aheterologous protein to Pichia methanolica. Optionally, a fusion proteinof the present invention may further include a third portion which mayinclude, for example, an immuglobulin moiety comprising at least oneconstant region, e.g., a human immunoglobulin Fc fragment, an affinitytag, a therapeutic agent, a detectable label, and the like.

The present invention also provides an isolated DNA molecule capable ofhybridizing to SEQ ID NO:1, or a complement thereof, under hybridizationconditions of 0.015 M NaCl/0.0015 M sodium citrate (SSC) and about 0.1percent sodium dodecyl sulfate (SDS) at about 50° C. to about 65° C. Thenucleic acid molecule may encode at least a portion of a polypeptide,such as a functional β-glucanase of Pichia methanolica.

The present invention also contemplates variant β-glucanase DNAmolecules that can be identified using two criteria: a determination ofthe similarity between the encoded polypeptide with the amino acidsequence of SEQ ID NO:2, and/or a hybridization assay, as describedabove. Such β-glucanase variants include nucleic acid molecules: (1)that hybridize with a nucleic acid molecule having the nucleotidesequence of SEQ ID NO:1 (or its complement) under stringent washingconditions, in which the wash stringency is equivalent to 0.5×-2×SSCwith 0.1% SDS at 55-65° C.; or (2) that encode a polypeptide having atleast 70%, at least 80%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or greater than 99% identity tothe amino acid sequence of SEQ ID NO:2. Alternatively, β-glucanasevariants can be characterized as nucleic acid molecules: (1) thathybridize with a nucleic acid molecule having the nucleotide sequence ofSEQ ID NO:1 (or its complement) under highly stringent washingconditions, in which the wash stringency is equivalent to 0.1×-0.2×SSCwith 0.1% SDS at 50-65° C.; and (2) that encode a polypeptide having atleast 70%, at least 80%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or greater than 99% sequenceidentity to the amino acid sequence of SEQ ID NO:2.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes).$\frac{{Total}\quad{number}\quad{of}\quad{identical}\quad{matches}}{\begin{matrix}\left\lbrack {{length}\quad{of}\quad{the}\quad{longer}\quad{sequence}\quad{plus}\quad{the}} \right. \\{{number}\quad{of}\quad{gaps}\quad{introduced}\quad{into}\quad{the}\quad{longer}} \\\left. {{sequence}\quad{in}\quad{order}\quad{to}\quad{align}\quad{the}\quad{two}\quad{sequences}} \right\rbrack\end{matrix}} \times 100$ TABLE 3 A R N D C Q E G H I L K M F P S T W YV A 4 R −1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 00 2 −4 2 5 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3−1 −3 −3 −4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2−1 −3 −2 5 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3−3 −1 0 0 −3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 10 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1−2 −1 1 5 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2−2 −3 −2 −1 −2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3−3 3 1 −2 1 −1 −2 −2 0 −3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant β-glucanase. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NO:2) and a testsequence that have either the highest density of identities (if the ktupvariable is 1) or pairs of identities (if ktup=2), without consideringconservative amino acid substitutions, insertions, or deletions. The tenregions with the highest density of identities are then rescored bycomparing the similarity of all paired amino acids using an amino acidsubstitution matrix, and the ends of the regions are “trimmed” toinclude only those residues that contribute to the highest score. Ifthere are several regions with scores greater than the “cutoff” value(calculated by a predetermined formula based upon the length of thesequence and the ktup value), then the trimmed initial regions areexamined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62. These parameters can be introducedinto a FASTA program by modifying the scoring matrix file (“SMATRIX”),as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdefault.

Variant β-glucanase polypeptides or polypeptides with substantiallysimilar sequence identity are characterized as having one or more aminoacid substitutions, deletions or additions. These changes are preferablyof a minor nature, that is conservative amino acid substitutions (asshown in Table 4 below) and other substitutions that do notsignificantly affect the folding or activity of the polypeptide; smalldeletions, typically of one to about 10 amino acids, preferably one toabout 5 amino acids; and amino- or carboxyl-terminal extensions, suchas, for instance, an amino-terminal methionine residue, a small linkerpeptide of up to about 5-20 residues, therapeutic agent, a detectablelabel, or an affinity tag. The present invention thus includespolypeptides of about 15-100 amino acid residues that comprise asequence that is at least 70%, at least 80%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, or greater than99% identical to the corresponding region of SEQ ID NO:2. Polypeptidescomprising affinity tags can further comprise a proteolytic cleavagesite between the β-glucanase polypeptide and the affinity tag. Preferredsuch sites include thrombin cleavage sites and factor Xa cleavage sites.Polypeptides of the present invention are preferably recombinantpolypeptides. In another aspect, the β-glucanase polypeptides of thepresent invention have at least 10, at least 15, at least 20, or atleast 25 contiguous amino acids. For example, a β-glucanase polypeptideof the present invention relates to a polypeptide having at least 10, atleast 15, at least 20, or at least 25 contiguous amino acids of SEQ IDNO:2. TABLE 4 Conservative amino acid substitutions Basic: argininelysine histidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

Determination of amino acid residues that comprise regions or domainsthat are critical to maintaining structural integrity can be determined.Within these regions one can determine specific residues that will bemore or less tolerant of change and maintain the overall tertiarystructure of the molecule. Methods for analyzing sequence structureinclude, but are not limited to, alignment of multiple sequences withhigh amino acid or nucleotide identity, secondary structurepropensities, binary patterns, complementary packing and buried polarinteractions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 andCordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). In general,when designing modifications to molecules or identifying specificfragments determination of structure will be accompanied by evaluatingactivity of modified molecules.

Amino acid sequence changes are made in β-glucanase polypeptides so asto minimize disruption of higher order structure essential to biologicalactivity. The effects of amino acid sequence changes can be predictedby, for example, computer modeling as disclosed above or determined byanalysis of crystal structure (see, e.g., Lapthorn et al., Nat. Struct.Biol. 2:266-268, 1995). Other techniques that are well known in the artcompare folding of a variant protein to a standard molecule (e.g., thenative protein). For example, comparison of the cysteine pattern in avariant and standard molecules can be made. Mass spectrometry andchemical modification using reduction and alkylation provide methods fordetermining cysteine residues which are associated with disulfide bondsor are free of such associations (Bean et al., Anal. Biochem.201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Pattersonet al., Anal. Chem. 66:3727-3732, 1994). It is generally believed thatif a modified molecule does not have the same cysteine pattern as thestandard molecule folding would be affected. Another well known andaccepted method for measuring folding is circular dichrosism (CD).Measuring and comparing the CD spectra generated by a modified moleculeand standard molecule is routine (Johnson, Proteins 7:205-214, 1990).Crystallography is another well known method for analyzing folding andstructure. Nuclear magnetic resonance (NMR), digestive peptide mappingand epitope mapping are also known methods for analyzing folding andstructurally similarities between proteins and polypeptides (Schaanan etal., Science 257:961-964, 1992).

A Hopp/Woods hydrophilicity profile of the β-glucanase protein sequenceas shown in SEQ ID NO:2 can be generated (Hopp et al., Proc. Natl. Acad.Sci., 78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 andTriquier et al., Protein Engineering 11:153-169, 1998). The profile isbased on a sliding six-residue window. Buried G, S, and T residues andexposed H, Y, and W residues were ignored.

Those skilled in the art will recognize that hydrophilicity orhydrophobicity will be taken into account when designing modificationsin the amino acid sequence of a β-glucanase polypeptide, so as not todisrupt the overall structural and biological profile. Of particularinterest for replacement are hydrophobic residues selected from thegroup consisting of Val, Leu and Ile or the group consisting of Met,Gly, Ser, Ala, Tyr and Trp. For example, residues tolerant ofsubstitution could include Val, Leu and Ile or the group consisting ofMet, Gly, Ser, Ala, Tyr and Trp residues as shown in SEQ ID NO:2.Conserved cysteine residues at positions within SEQ ID NO:2 will berelatively intolerant of substitution.

Using methods such as “FASTA” analysis described previously, regions ofhigh similarity are identified within a family of proteins and used toanalyze amino acid sequence for conserved regions. An alternativeapproach to identifying a variant β-glucanase polynucleotide on thebasis of structure is to determine whether a nucleic acid moleculeencoding a potential variant β-glucanase gene can hybridize to a nucleicacid molecule having the nucleotide sequence of SEQ ID NO:1, asdiscussed above.

Other methods of identifying essential amino acids in the polypeptidesof the present invention are procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Natl. Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalor biochemical activity as disclosed below to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., J. Biol. Chem. 271:4699 (1996).

The present invention also provides a fusion protein comprising a firstportion and a second portion, wherein the first portion and the secondportion are joined by a peptide bond, wherein the first portioncomprises a functional β-glucanase, such as a polypeptide having atleast 95 percent sequence identity with SEQ ID NO:2 or comprising SEQ IDNO:2, and the second portion comprises a protein of interest, such as aheterologous protein. The fusion protein may optionally comprise a thirdportion, such as an affinity tag, a therapeutic agent, detectable labeland the like. The present invention also provides DNA molecules encodingthe fusion proteins of the present invention.

The present invention also provides DNA constructs comprising thefollowing operably linked elements: a first DNA segment comprising atranscription promoter of Pichia methanolica, a second DNA segmentcomprising a nucleotide sequence encoding a polypeptide of SEQ ID NO:2or a polypeptide having 95 percent sequence identity with SEQ ID NO:2, athird DNA segment encoding a protein of interest, and a fourth DNAsegment comprising a transcription terminator of Pichia methanolica. Thefirst DNA segment may be a transcription promoter such as, for instance,glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1),glyceraldehyde-3-phosphate dehydrogenase 2 (GAP2), alcohol utilizationgene 1 (AUG1), alcohol utilization gene 2 (AUG2), and other Pichiamethanolica promoters. The second DNA segment is a functional Pichiamethanolica β-glucanse gene, e.g., SEQ ID NO:1. The third DNA segmentpreferably encodes a heterologous protein. The fourth DNA segmentincludes a Pichia methanolica transcription terminator, such as, forinstance, GAP1, GAP2, AUG1, AUG2, and other Pichia methanolicaterminators.

A DNA construct of the present invention may further comprise aselectable marker, e.g., ADE2 gene. In addition, a DNA construct of thepresent invention may further comprise a Pichia methanolica origin ofreplication or an additional origin of replication from anotherorganism, e.g., E. coli, Chinese hamster overy (CHO) cells, baby hamsterkidney (BHK) cells, and the like. For example, a DNA construct of thepresent invention can be amplified, for instance, in E. coli thenshuttled to a host cell, such as CHO cells, for protein expression.

A DNA construct of the present invention may further include a fifthoperably linked DNA segment wherein the fifth DNA segment comprises animmunoglobulin moiety comprising at least one constant region, forexample, a human immunoglobulin Fc fragment, an affinity tag, atherapeutic agent and/or a detectable label.

Cultured mammalian cells are suitable hosts for DNA constructs of thepresent invention. Methods for introducing exogenous DNA into mammalianhost cells include calcium phosphate-mediated transfection (Wigler etal., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973),electroporation (Neumann et al., EMBO J. 1:841-5, 1982), DEAE-dextranmediated transfection (Ausubel et al., ibid.), and liposome-mediatedtransfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al.,Focus 15:80, 1993, and viral vectors (Miller and Rosman, BioTechniques7:980-90, 1989; Wang and Finer, Nature Med. 2:714-6, 1996). Theproduction of recombinant polypeptides in cultured mammalian cells isdisclosed, for example, by Levinson et al., U.S. Pat. No. 4,713,339;Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No.4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitable culturedmammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No.CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293(ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) andChinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines.Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Manassas, Va. In general, strong transcription promoters are preferred,such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat.No. 4,956,288. Other suitable promoters include those frommetallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and theadenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g., hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plantcells, insect cells and avian cells. The use of Agrobacterium rhizogenesas a vector for expressing genes in plant cells has been reviewed bySinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication No. WO94/06463. Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV). See, King, L. A. and Possee, R. D., The Baculovirus ExpressionSystem: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. etal., Baculovirus Expression Vectors: A Laboratory Manual, New York,Oxford University Press., 1994; and, Richardson, C. D., Ed., BaculovirusExpression Protocols. Methods in Molecular Biology, Totowa, N.J., HumanaPress, 1995. The second method of making recombinant baculovirusutilizes a transposon-based system described by Luckow (Luckow, V. A, etal., J Virol 67:4566-79, 1993). This system is sold in the Bac-to-Backit (Life Technologies, Rockville, Md.). This system utilizes a transfervector, pFastBac1™ (Life Technologies) containing a Tn7 transposon tomove the DNA encoding the β-glucanase fusion protein into a baculovirusgenome maintained in E. coli as a large plasmid called a “bacmid.” ThepFastBac1™ transfer vector utilizes the AcNPV polyhedrin promoter todrive the expression of the gene of interest. However, pFastBac1™ can bemodified to a considerable degree. The polyhedrin promoter can beremoved and substituted with the baculovirus basic protein promoter(also known as Pcor, p6.9 or MP promoter) which is expressed earlier inthe baculovirus infection, and has been shown to be advantageous forexpressing secreted proteins. See, Hill-Perkins, M. S. and Possee, R.D., J. Gen. Virol. 71:971-6, 1990; Bonning, B. C. et al., J. Gen. Virol.75:1551-6, 1994; and, Chazenbalk, G. D., and Rapoport, B., J. Biol.Chem. 270:1543-9, 1995. In such transfer vector constructs, a short orlong version of the basic protein promoter can be used.

Using techniques known in the art, a transfer vector containingβ-glucanase fusion protein is transformed into E. Coli, and screened forbacmids which contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is isolated, using common techniques, and used to transfectSpodoptera frugiperda cells, e.g., Sf9 cells. Recombinant virus thatexpresses β-glucanase fusion protein is subsequently produced.Recombinant viral stocks are made by methods commonly used the art.

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein Saccharomyces cerevisiae, is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia guillermondii and Candida maltosa are known in the art.See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-65, 1986and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349.Methods for transforming Acremonium chrysogenum are disclosed by Suminoet al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora aredisclosed by Lambowitz, U.S. Pat. No. 4,486,533.

Heterologous or exogenous DNA can also be introduced into P. methanolicacells, another useful yeast host cell, by any of several known methods,including lithium transformation (Hiep et al., Yeast 9:1189-1197, 1993;Tarutina and Tolstorukov, Abst. of the 15th International SpecializedSymposium on Yeasts, Riga (USSR), 1991, 137; Ito et al., J. Bacteriol.153:163, 1983; Bogdanova et al., Yeast 11:343, 1995), spheroplasttransformation (Beggs, Nature 275:104, 1978; Hinnen et al., Proc. Natl.Acad. Sci. USA 75:1929, 1978; Cregg et al., Mol. Cell. Biol. 5:3376,1985), freeze-thaw polyethylene glycol transformation (Pichia ExpressionKit Instruction Manual, Invitrogen Corp., San Diego, Calif., Cat. No.K1710-01), or electroporation, the latter being preferred.Electroporation is the process of using a pulsed electric field totransiently permeabilize cell membranes, allowing macromolecules, suchas DNA, to pass into cells. Electroporation has been described for usewith mammalian (e.g., Neumann et al., EMBO J. 1:841-845, 1982) andfungal (e.g., Meilhoc et al., Biotechnology 8:223-227, 1990) host cells.However, the actual mechanism by which DNA is transferred into the cellsis not well understood. For transformation of P. methanolica, it hasbeen found that electroporation is surprisingly efficient when the cellsare exposed to an exponentially decaying, pulsed electric field having afield strength of from 2.5 to 4.5 kV/cm and a time constant (τ) of from1 to 40 milliseconds. The time constant τ is defined as the timerequired for the initial peak voltage V₀ to drop to a value of V₀/e. Thetime constant can be calculated as the product of the total resistanceand capacitance of the pulse circuit, i.e., τ=R×C. Typically, resistanceand capacitance are either preset or may be selected by the user,depending on the electroporation equipment selected. In any event, theequipment is configured in accordance with the manufacturer'sinstructions to provide field strength and decay parameters as disclosedabove. Electroporation equipment is available from commercial suppliers(e.g., BioRad Laboratories, Hercules, Calif.).

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells, for example, are cultured in a medium comprising adequate sourcesof carbon, nitrogen and trace nutrients at a temperature of about 25° C.to 35° C. Liquid cultures are provided with sufficient aeration byconventional means, such as shaking of small flasks or sparging offermentors. A preferred culture medium for P. methanolica is YEPD (2%D-glucose, 2% Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1%Bacto™ yeast extract (Difco Laboratories), 0.004% adenine and 0.006%L-leucine).

DNA molecules for use in transforming P. methanolica will commonly beprepared as double-stranded, circular plasmids, which are preferablylinearized prior to transformation. For polypeptide or proteinproduction, the DNA molecules will include, in addition to theselectable marker disclosed herein, an expression cassette comprising atranscription promoter, a functional glucanase gene, a DNA segment(e.g., a cDNA) encoding the polypeptide or protein of interest, and atranscription terminator. These elements are operably linked to providefor transcription of the DNA segment of interest. It is preferred thatthe promoter and terminator be that of a P. methanolica gene. Usefulpromoters include those from constitutive and methanol-induciblepromoters. Promoter sequences are generally contained within 1.5 kbupstream of the coding sequence of a gene, often within 1 kb or less. Ingeneral, regulated promoters are larger than constitutive promoters duethe presence of regulatory elements. Methanol-inducible promoters, whichinclude both positive and negative regulatory elements, may extend morethan 1 kb upstream from the initiation ATG. Promoters are identified byfunction and can be cloned according to known methods.

A methanol-inducible promoter that may be used is that of a P.methanolica alcohol utilization gene. A representative coding strandsequence of one such gene is AUG1 (Raymond et al., U.S. Pat. No.6,153,424). P. methanolica contains a second alcohol utilization gene,AUG2, the promoter of which can be used within the present invention(Raymond et al., U.S. Pat. No. 6,153,424). Other useful promotersinclude those of the dihydroxyacetone synthase (DHAS), formatedehydrogenase (FMD), and catalase (CAT) genes. Genes encoding theseenzymes from other species have been described, and their sequences areavailable (e.g., Janowicz et al., Nuc. Acids Res. 13:2043, 1985;Hollenberg and Janowicz, EPO publication 0 299 108; Didion andRoggenkamp, FEBS Lett. 303:113, 1992). Genes encoding these proteins canbe cloned by using the known sequences as probes, or by aligning knownsequences, designing primers based on the alignment, and amplifying P.methanolica DNA by the polymerase chain reaction (PCR).

Constitutive promoters are those that are not activated or inactivatedby environmental conditions; they are always transcriptionally active.Preferred constitutive promoters for use within the present inventioninclude those from glyceraldehyde-3-phosphate dehydrogenase (asdescribed herein), triose phosphate isomerase, and phosphoglyceratekinase genes of P. methanolica. These genes can be cloned as disclosedabove or by complementation in a host cell, such as a Saccharomycescerevisiae cell, having a mutation in the counterpart gene. Mutants ofthis type are well known in the art. See, for example, Kawasaki andFraenkel, Biochem. Biophys. Res. Comm. 108:1107-1112, 1982; McKnight etal., Cell 46:143-147, 1986; Aguilera and Zimmermann, Mol. Gen. Genet.202:83-89, 1986.

The DNA molecule of the present invention can comprise a Pichiamethanolica glyceraldehydes-3-phosphate dehydrogenase-1 (GAPDH-1)promoter and terminator (SEQ ID NO:5) (Raymond et al., WO 00/78978), andPichia methanolica glyceraldehydes-3-phosphate dehydrogenase-2 (GAPDH-2)promoter and terminator (SEQ ID NO:6) (Raymond, U.S. Pat. Nos. 6,348,331and 6,440,720). For large scale, industrial processes where it isdesirable to minimize the use of methanol, host cells may be used thathave a genetic defect in a gene required for methanol utilization. Suchgenes include alcohol oxidase genes AUG1 and AUG2 (Zamost, B., U.S. Pat.No. 6,258,559), as well as genes encoding catalase, formaldehydedehydrogenase, formate dehydrogenase, dihydroxyacetone synthase,dihydroxyacetone kinase, fructose 1,6-bisphosphate aldolase, andfructose 1,6-bisphosphatase. It is particularly advantageous to usecells in which both alcohol oxidase genes (AUG1 and AUG2) are deleted.Methods for producing Pichia methanolica strains that have a defect inAUG1, AUG2, or both AUG1 and AUG2 genes are described by Raymond et al.,Yeast 14:11 (1998), by Raymond, U.S. Pat. No. 5,716,808, and by Raymondet al., U.S. Pat. No. 5,736,383.

The sequence of a DNA molecule comprising a P. methanolicaglyceraldehyde-3-phosphate dehydrogenase-1 (GAPDH-1) gene promoter,coding region, and terminator is shown in SEQ ID NO:5. The gene has beendesignated GAP1. Those skilled in the art will recognize that SEQ IDNO:5 represents a single allele of the P. methanolica GAP1 gene and thatother functional alleles (allelic variants) are likely to exist, andthat allelic variation may include nucleotide changes in the promoterregion, coding region, or terminator region.

Within SEQ ID NO:5, the GAP1 open reading frame begins with themethionine codon (ATG) at nucleotides 1733-1735. The transcriptionpromoter is located upstream of the ATG. Gene expression experimentsshowed that a functional promoter was contained within the ca. 900nucleotide 5′-flanking region of the GAP1 gene. Analysis of thispromoter sequence revealed the presence of a number of sequenceshomologous to Saccharomyces cerevisiae promoter elements. Thesesequences include a concensus TATAAA box at nucleotides 1584 to 1591, aconsensus Raplp binding site (Graham and Chambers, Nuc. Acids Res.22:124-130, 1994) at nucleotides 1355 to 1367, and potential Gcrlpbinding sites (Shore, Trends Genet. 10:408-412, 1994) at nucleotides1225 to 1229, 1286 to 1290, 1295 to 1299, 1313 to 1317, 1351 to 1354,1370 to 1374, 1389 to 1393, and 1457 to 1461. While not wishing to bebound by theory, it is believed that these sequences may performfunctions similar to those of their counterparts in the S. cerevisiaeTDH3 promoter (Bitter et al., Mol. Gen. Genet. 231:22-32, 1991), thatis, they may bind the homologous transcription regulatory elements.Mutation of the region around the consensus Gcrlp binding site in the P.methanolica GAP1 promoter has been found to destroy promoter activity.

Preferred portions of the sequence shown in SEQ ID NO:5 for use withinthe present invention as transcription promoters include segmentscomprising at least 900 contiguous nucleotides of the 5′ non-codingregion of SEQ ID NO:5, and preferably comprising nucleotide 810 tonucleotide 1724 of the sequence shown in SEQ ID NO:5. Those skilled inthe art will recognize that longer portions of the 5′ non-coding regionof the P. methanolica GAP1 gene can also be used. Promoter sequences ofthe present invention can thus include the sequence of SEQ ID NO:5through nucleotide 1732 in the 3′ direction and can extend to or beyondnucleotide 232 in the 5′ direction. For convenience and ease ofmanipulation, the promoter used within an expression DNA construct willgenerally not exceed 1.5 kb in length, and will often not exceed 1.0 kbin length.

As disclosed in more detail in the examples that follow, the sequence ofSEQ ID NO:5 from nucleotide 810 to 1724 provides a functionaltranscription promoter. However, additional nucleotides can be removedfrom either or both ends of this sequence and the resulting sequencetested for promoter function by joining it to a sequence encoding aprotein, preferably a protein for which a convenient assay is readilyavailable.

Within the present invention it is preferred that the GAP1 promoter besubstantially free of GAP1 gene coding sequence, which begins withnucleotide 1733 in SEQ ID NO:1. As used herein, the term “substantiallyfree of GAP1 gene coding sequence” means that the promoter DNA includesnot more than 15 nucleotides of the GAP1 coding sequences, preferablynot more than 10 nucleotides, and more preferably not more than 3nucleotides. Within one embodiment of the invention, the GAP1 promoteris provided free of coding sequence of the P. methanolica GAP1 gene.However, those skilled in the art will recognize that a GAP1 genefragment that includes the initiation ATG (nucleotides 1733 to 1735) ofSEQ ID NO:5 can be operably linked to a heterologous coding sequencethat lacks an ATG, with the GAP1 ATG providing for initiation oftranslation of the heterologous sequence. Those skilled in the art willfurther recognize that additional GAP1 coding sequences can also beincluded, whereby a fusion protein comprising GAP1 and heterologousamino acid sequences is produced. Such a fusion protein may comprise acleavage site to facilitate separation of the GAP1 and heterologoussequences subsequent to translation.

In addition to the GAP1 promoter sequence, the present invention alsoprovides transcription terminator sequences derived from the 3′non-coding region of the P. methanolica GAP1 gene. A consensustranscription termination sequence (Chen and Moore, Mol. Cell. Biol.12:3470-3481, 1992) is at nucleotides 2774 to 2787 of SEQ ID NO:5.Within the present invention, there are thus provided transcriptionterminator gene segments of at least about 60 bp in length. Longersegments, for example at least 90 bp in length or about 200 bp inlength, will often be used. These segments comprise the terminationsequence disclosed above, and may have as their 5′ termini nucleotide2735 of SEQ ID NO:5. Those skilled in the art will recognize, however,that the transcription terminator segment that is provided in an DNAconstruct can include at its 5′ terminus the TAA translation terminationcodon at nucleotides 2732-2734 of SEQ ID NO:5 to permit the insertion ofcoding sequences that lack a termination codon.

The present invention also provides a DNA molecule comprising a Pichiamethanolica glyceraldehyde-3-phosphate dehydrogenase-2 (GAPDH-2) genepromoter, coding region, and terminator as shown in SEQ ID NO:6. Thegene has been designated GAP2. Those skilled in the art will recognizethat SEQ ID NO:6 represents a single allele of the P. methanolica GAP2gene and that other functional alleles (allelic variants) are likely toexist, and that allelic variation may include nucleotide changes in thepromoter region, coding region, or terminator region.

Within SEQ ID NO:6, the GAP2 open reading frame begins with themethionine codon (ATG) at nucleotides 1093-1095. The transcriptionpromoter is located upstream of the ATG. Gene expression experimentsshowed that a functional promoter was contained within the ca. 1000nucleotide 5′-flanking region of the GAP2 gene.

Preferred portions of the sequence shown in SEQ ID NO:6 for use withinthe present invention as transcription promoters include segmentscomprising at least 900 contiguous nucleotides of the 5′ non-codingregion of SEQ ID NO:6, and preferably comprising nucleotide 93 tonucleotide 1080 of the sequence shown in SEQ ID NO:6. Those skilled inthe art will recognize that longer portions of the 5′ non-coding regionof the P. methanolica GAP2 gene can also be used. Promoter sequences ofthe present invention can thus include the sequence of SEQ ID NO:6through nucleotide 1092 in the 3′ direction and can extend to or beyondnucleotide 1 in the 5′ direction. In general, the promoter used withinan expression DNA construct will not exceed 1.5 kb in length, and willpreferably not exceed 1.0 kb in length. In addition to these promoterfragments, the invention also provides isolated DNA molecules of up toabout 3300 bp, as well as isolated DNA molecules of up to 5000 bp,wherein said molecules comprise the P. methanolica GAP2 promotersequence.

Within the present invention it is preferred that the GAP2 promoter besubstantially free of GAP2 gene coding sequence, which begins withnucleotide 1093 in SEQ ID NO:6. As used herein, “substantially free” ofGAP2 gene coding sequence means that the promoter DNA includes not morethan 15 nucleotides of the GAP2 coding sequence, preferably not morethan 10 nucleotides, and more preferably not more than 3 nucleotides.Within a preferred embodiment of the invention, the GAP2 promoter isprovided free of coding sequence of the P. methanolica GAP2 gene.However, those skilled in the art will recognize that a GAP2 genefragment that includes the initiation ATG (nucleotides 1093 to 1095) ofSEQ ID NO:6 can be operably linked to a heterologous coding sequencethat lacks an ATG, with the GAP2 ATG providing for initiation oftranslation of the heterologous sequence. Those skilled in the art willfurther recognize that additional GAP2 coding sequences can also beincluded, whereby a fusion protein comprising GAP2 and heterologousamino acid sequences is produced. Such a fusion protein may comprise acleavage site to facilitate separation of the GAP2 and heterologoussequences subsequent to translation.

In addition to the GAP2 promoter sequence, the present invention alsoprovides transcription terminator sequences derived from the 3′non-coding region of the P. methanolica GAP2 gene. A consensustranscription termination sequence (Chen and Moore, Mol. Cell. Biol.12:3470-3481, 1992) is at nucleotides 2136 to 2145 of SEQ ID NO:6.Within the present invention, there are thus provided transcriptionterminator gene segments of at least about 50 bp, preferably at least 60bp, more preferably at least 90 bp, still more preferably about 200 bpin length. The terminator segments of the present invention may comprise500-1000 nucleotides of the 3′ non-coding region of SEQ ID NO:6. Thesesegments comprise the termination sequence disclosed above, andpreferably have as their 5′ termini nucleotide 2095 of SEQ ID NO:6.Those skilled in the art will recognize, however, that the transcriptionterminator segment that is provided in an expression vector can includeat its 5′ terminus the TAA translation termination codon at nucleotides2092-2094 of SEQ ID NO:6 to permit the insertion of coding sequencesthat lack a termination codon.

A DNA construct of the present invention may further include aselectable marker. Expression vectors or DNA constructs of the presentinvention further comprise a selectable marker to permit identificationand selection of P. methanolica cells containing the vector. Selectablemarkers provide for a growth advantage of cells containing them. Thegeneral principles of selection are well known in the art. Theselectable marker is preferably a P. methanolica gene. Commonly usedselectable markers are genes that encode enzymes required for thesynthesis of amino acids or nucleotides. Cells having mutations in thesegenes cannot grow in media lacking the specific amino acid or nucleotideunless the mutation is complemented by the selectable marker. Use ofsuch “selective” culture media ensures the stable maintenance of theheterologous DNA within the host cell. A selectable marker of thepresent invention for use in P. methanolica may include, for instance, aP. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazolecarboxylase (AIRC; EC 4.1.1.21). See, Raymond, U.S. Pat. No. 5,736,383.The ADE2 gene, when transformed into an ade2 host cell, allows the cellto grow in the absence of adenine. The coding strand of a representativeP. methanolica ADE2 gene sequence is shown in SEQ ID NO:4. The sequenceillustrated includes 1006 nucleotides of 5′ non-coding sequence and 442nucleotides of 3′ non-coding sequence, with the initiation ATG codon atnucleotides 1007-1009. Within a preferred embodiment of the invention, aDNA segment comprising nucleotides 407-2851 is used as a selectablemarker, although longer or shorter segments could be used as long as thecoding portion is operably linked to promoter and terminator sequences.In the alternative, a dominant selectable marker, which provides agrowth advantage to wild-type cells, may be used. Typical dominantselectable markers are genes that provide resistance to antibiotics,such as neomycin-type antibiotics (e.g., G418), hygromycin B, andbleomycin/phleomycin-type antibiotics (e.g., Zeocin™; available fromInvitrogen Corporation, San Diego, Calif.). A preferred dominantselectable marker for use in P. methanolica is the Sh bla gene, whichinhibits the activity of Zeocin™.

The present invention also provides a Pichia methanolica cell containinga DNA construct as described herein. The DNA construct may begenomically integrated into the Pichia methanolica genome with one ormore copies. The Pichia methanolica cell may have a functionallydeficient vacuolar proteinease A and/or vacuolar proteinase B. ThePichia methanolica cell may have a functionally deficient AUG1 and/orAUG2 gene.

The present invention also provides a method of producing a protein ofinterest comprising: culturing a cell of the present invention whereinthe cell containing a DNA construct of the present invention wherein thethird DNA segment is expressed and the protein of interest is produced,and recovering the protein of interest. Preferably, the protein ofinterest is heterologous or foreign to Pichia methanolica.

Techniques for manipulating cloned DNA molecules and introducingexogenous DNA into a variety of host cells are well known in the art andare disclosed by, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989; Murray, ed., Gene Transfer and ExpressionProtocols, Humana Press, Clifton, N.J., 1991; Glick and Pasternak,Molecular Biotechnology: Principles and Applications of Recombinant DNA,ASM Press, Washington, D.C., 1994; Ausubel et al. (eds.), ShortProtocols in Molecular Biology, 3rd edition, John Wiley and Sons, Inc.,NY, 1995; Wu et al., Methods in Gene Biotechnology, CRC Press, New York,1997. DNA vectors, including expression vectors, commonly contain aselectable marker and origin of replication that function in a bacterialhost (e.g., E. coli) to permit the replication and amplification of thevector in a prokaryotic host. If desired, these prokaryotic elements canbe removed from a vector before it is introduced into an alternativehost. For example, such prokaryotic sequences can be removed bylinearization of the vector prior to its introduction into a P.methanolica host cell.

Within other embodiments of the invention, DNA constructs are providedthat comprise a DNA segment comprising a portion of SEQ ID NO:6 that isa functional transcription terminator operably linked to a functionalβ-glucanase gene of the present invention, and an additional DNA segmentencoding a protein of interest. Within one embodiment, the GAP2 promoterand terminator sequences of the present invention are used incombination, wherein both are operably linked to a functionalβ-glucanase gene and a DNA segment encoding a protein of interest withina DNA construct.

The use of P. methanolica cells as a host for the production ofrecombinant proteins is disclosed in U.S. Pat. Nos. 5,955,349,5,888,768, 6,001,597, 5,965,389, 5,736,383, 5,854,039, 5,716,808,5,736,383, 5,854,039, and 5,736,383. DNA constructs, e.g., expressionvectors, for use in transforming P. methanolica will commonly beprepared as double-stranded, circular plasmids, which are preferablylinearized prior to transformation. To facilitate integration of theexpression vector DNA into the host chromosome, it is preferred to havethe entire expression segment of the plasmid flanked at both ends byhost DNA sequences (e.g., AUG1 3′ sequences). Electroporation is used tofacilitate the introduction of a plasmid containing DNA encoding apolypeptide of interest into P. methanolica cells. It is preferred totransform P. methanolica cells by electroporation using an exponentiallydecaying, pulsed electric field having a field strength of from 2.5 to4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (τ) of from1 to 40 milliseconds, most preferably about 20 milliseconds.

Integrative transformants are preferred for use in protein productionprocesses. Such cells can be propagated without continuous selectivepressure because DNA is rarely lost from the genome. Integration of DNAinto the host chromosome can be confirmed by Southern blot analysis.Briefly, transformed and untransformed host DNA is digested withrestriction endonucleases, separated by electrophoresis, blotted to asupport membrane, and probed with appropriate host DNA segments.Differences in the patterns of fragments seen in untransformed andtransformed cells are indicative of integrative transformation.Restriction enzymes and probes can be selected to identify transformingDNA segments (e.g., promoter, terminator, heterologous DNA, andselectable marker sequences) from among the genomic fragments.

Differences in expression levels of heterologous proteins can resultfrom such factors as the site of integration and copy number of theexpression cassette among individual isolates. It is thereforeadvantageous to screen a number of isolates for expression level priorto selecting a production strain. Isolates exhibiting a high expressionlevel will commonly contain multiple integrated copies of the desiredexpression cassette. A variety of suitable screening methods areavailable. For example, transformant colonies are grown on plates thatare overlayed with membranes (e.g., nitrocellulose) that bind protein.Proteins are released from the cells by secretion or following lysis,and bind to the membrane. Bound protein can then be assayed using knownmethods, including immunoassays. More accurate analysis of expressionlevels can be obtained by culturing cells in liquid media and analyzingconditioned media or cell lysates, as appropriate. Methods forconcentrating and purifying proteins from media and lysates will bedetermined in part by the protein of interest. Such methods are readilyselected and practiced by the skilled practitioner.

For production of secreted proteins, host cells having functionaldeficiencies in the vacuolar proteases proteinase A, which is encoded bythe PEP4 gene, and proteinase B, which is encoded by the PRB1 gene, arepreferred in order to minimize spurious proteolysis (Raymond et al.,U.S. Pat. No. 6,153,424). Vacuolar protease activity (and thereforevacuolar protease deficiency) is measured using any of several knownassays. Preferred assays are those developed for Saccharomycescerevisiae and disclosed by Jones, Methods Enzymol. 194:428-453, 1991. Apreferred such assay is the APNE overlay assay, which detects activityof carboxypeptidase Y (CpY). See, Wolf and Fink, J. Bact. 123:1150-1156,1975. Because the zymogen (pro)CpY is activated by proteinase A andproteinase B, the APNE assay is indicative of vacuolar protease activityin general. The APNE overlay assay detects the carboxypeptidaseY-mediated release of β-naphthol fromN-acetyl-phenylalanine-β-naphthyl-ester (APNE), which results in theformation of an isoluble red dye by the reaction of the β-naphthol withthe diazonium salt Fast Garnet GBC. Cells growing on assay plates (YEPDplates are preferred) at room temperature are overlayed with 8 ml RxM.RxM is prepared by combining 0.175 g agar, 17.5 ml H₂O, and 5 ml 1 MTris-HCl pH 7.4, microwaving the mixture to dissolve the agar, coolingto ˜55° C., adding 2.5 ml freshly made APNE (2 mg/ml indimethylformamide) (Sigma Chemical Co., St. Louis, Mo.), and,immediately before assay, 20 mg Fast Garnet GBC salt (Sigma ChemicalCo.). The overlay is allowed to solidify, and color development isobserved. Wild-type colonies are red, whereas CPY deletion strains arewhite. Carboxypeptidase Y activity can also be detected by the welltest, in which cells are distributed into wells of a microtiter testplate and incubated in the presence of N-benzoyl-L-tyrosinep-nitroanilide (BTPNA) and dimethylformamide. The cells arepermeabilized by the dimethylformamide, and CpY in the cells cleaves theamide bond in the BTPNA to give the yellow product p-nitroaniline.Assays for CpY will detect any mutation that reduces protease activityso long as that activity ultimately results in the reduction of CpYactivity.

P. methanolica cells are cultured in a medium comprising adequatesources of carbon, nitrogen and trace nutrients at a temperature ofabout 25° C. to 35° C. Liquid cultures are provided with sufficientaeration by conventional means, such as shaking of small flasks orsparging of fermentors. A preferred culture medium for P. methanolica isYEPD (2% D-glucose, 2% Bacto™ Peptone (Difco Laboratories, Detroit,Mich.), 1% Bacto™ yeast extract (Difco Laboratories), 0.004% adenine,0.006% L-leucine).

For large-scale culture, one to two colonies of a P. methanolica straincan be picked from a fresh agar plate (e.g., YEPD agar) and suspended in250 ml of YEPD broth contained in a two-liter baffled shake flask. Theculture is grown for 16 to 24 hours at 30° C. and 250 rpm shaking speed.Approximately 50 to 80 milliliters of inoculum are used per literstarting fermentor volume (5-8% v/v inoculum).

A preferred fermentation medium is a soluble medium comprising glucoseas a carbon source, inorganic ammonia, potassium, phosphate, iron, andcitric acid. As used herein, a “soluble medium” is a medium that doesnot contain visible precipitation. Preferably, the medium lacksphosphate glass (sodium hexametaphosphate). A preferred medium isprepared in deionized water and does not contain calcium sulfate. As aminimal medium, it is preferred that the medium lacks polypeptides orpeptides, such as yeast extracts. However, acid hydrolyzed casein (e.g.,casamino acids or amicase) can be added to the medium if desired. Anillustrative fermentation medium is prepared by mixing the followingcompounds: (NH₄)₂SO₄ (11.5 grams/liter), K₂HPO₄ (2.60 grams/liter),KH₂PO₄ (9.50 grams/liter), FeSO₄.7H₂O (0.40 grams/liter), and citricacid (1.00 gram/liter). After adding distilled, deionized water to oneliter, the solution is sterilized by autoclaving, allowed to cool, andthen supplemented with the following: 60% (w/v) glucose solution (47.5milliliters/liter), 10× trace metals solution (20.0 milliliters/liter),1 M MgSO₄ (20.0 milliliters/liter), and vitamin stock solution (2.00milliliters/liter). The 10× trace metals solution contains FeSO₄.7H₂O(100 mM), CuSO₄.5H₂O (2 mM), ZnSO₄.7H₂O (8 mM), MnSO₄.H₂O (8 mM),CoCl₂.6H₂O (2 mM), Na₂MoO₄.2H₂O (1 mM), H₃BO₃ (8 mM), KI (0.5 mM),NiSO₄.6H₂O (1 mM), thiamine (0.50 grams/liter), and biotin (5.00milligrams/liter). The vitamin stock solution contains inositol (47.00grams/liter), pantothenic acid (23.00 grams/liter), pyrodoxine (1.20grams/liter), thiamine (5.00 grams/liter), and biotin (0.10 gram/liter).Those of skill in the art can vary these particular ingredients andamounts. For example, ammonium sulfate can be substituted with ammoniumchloride, or the amount of ammonium sulfate can be varied, for example,from about 11 to about 22 grams/liter.

After addition of trace metals and vitamins, the pH of the medium istypically adjusted to pH 4.5 by addition of 10% H₃PO₄. Generally, about10 milliliters/liter are added, and no additional acid addition will berequired. During fermentation, the pH is maintained between about 3.5 toabout 5.5, or about 4.0 to about 5.0, depending on protein produced, byaddition of 5 N NH₄OH.

An illustrative fermentor is a BIOFLO 3000 fermentor system (NewBrunswick Scientific Company, Inc.; Edison, N.J.). This fermentor systemcan handle either a six-liter or a fourteen-liter fermentor vessel.Fermentations performed with the six-liter vessel are prepared withthree liters of medium, whereas fermentations performed with thefourteen-liter vessel are prepared with six liters of medium. Thefermentor vessel operating temperature is typically set to 30° C. forthe course of the fermentation, although the temperature can rangebetween 27-31° C. depending on the protein expressed. The fermentationis initiated in a batch mode. The glucose initially present is oftenused by approximately 10 hours elapsed fermentation time (EFT), at whichtime a glucose feed can be initiated to increase the cell mass. Anillustrative glucose feed contains 900 milliliters of 60% (w/v) glucose,60 milliliters of 50% (w/v) (NH₄)₂SO₄, 60 milliliters of 10× tracemetals solution, and 30 milliliters of 1 M MgSO₄ . Pichia methanolicafermentation is robust and requires high agitation, aeration, and oxygensparging to maintain the percentage dissolved oxygen saturation above30%. The percentage dissolved oxygen should not drop below 15% foroptimal expression and growth. The biomass typically reaches about 30 toabout 80 grams dry cell weight per liter at 48 hours EFT.

Proteins produced according to the present invention are recovered fromthe host cells using conventional methods. Secreted proteins arerecovered from the conditioned culture medium using standard methods,also selected for the particular protein. See, in general, Scopes,Protein Purification: Principles and Practice, Springer-Verlag, NewYork, 1994.

The materials and methods of the present invention can be used toproduce proteins of research, industrial, or pharmaceutical interest.Such proteins include enzymes, such as lipases, cellulases, andproteases; antibodies and fragments thereof; enzyme inhibitors,including protease inhibitors; growth factors such as platelet derivedgrowth factor (PDGF), fibroblast growth factors (FGF), epidermal growthfactor (EGF), vascular endothelial growth factors (VEGFs); glutamic aciddecarboxylase (GAD); cytokines, such as erythropoietin, thrombopoietin,colony stimulating factors, interleukins, and interleukin antagonist;hormones, such as insulin, proinsulin, leptin, and glucagon; adipocytecomplement related proteins, such as zsig37, zsig39, zacrp8 and thelike; and receptors, including growth factor receptors, which can beexpressed in truncated form (“soluble receptors”) or as fusion proteinswith, for example, immunoglobulin constant region sequences. DNAsencoding these and other proteins are known in the art. See, forexample, U.S. Pat. Nos. 4,889,919; 5,219,759; 4,868,119; 4,968,607;4,599,311; 4,784,950; 5,792,850; 5,827,734; 4,703,008; 4,431,740;4,762,791; 6,265,544; 6,566,499; 6,197,930; 6,482,612; and WIPOPublications WO 95/21920 and WO 96/22308.

It is particularly preferred to use the present invention to produceunglycosylated pharmaceutical proteins. Yeast cells, including P.methanolica cells, produce glycoproteins with carbohydrate chains thatdiffer from their mammalian counterparts. Mammalian glycoproteinsproduced in yeast cells may therefore be regarded as “foreign” whenintroduced into a mammal, and may exhibit, for example, differentpharmacokinetics than their naturally glycosylated counterparts.

The present invention also provides antibodies to polypeptides of thepresent invention. Antibodies to β-glucanase can be obtained, forexample, using as an antigen the product of β-glucanase expressionvector or β-glucanase isolated from a natural source. Particularlyuseful anti-β-glucanase antibodies “bind specifically” with β-glucanase.Antibodies are considered to be specifically binding if the antibodiesexhibit at least one of the following two properties: (1) antibodiesbind to β-glucanase with a threshold level of binding activity, and (2)antibodies do not significantly cross-react with polypeptides related toβ-glucanase.

With regard to the first characteristic, antibodies specifically bind ifthey bind to a β-glucanase polypeptide, peptide or epitope with abinding affinity (Ka) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ orgreater, more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹or greater. The binding affinity of an antibody can be readilydetermined by one of ordinary skill in the art, for example, byScatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660 (1949)). Withregard to the second characteristic, antibodies do not significantlycross-react with related polypeptide molecules, for example, if theydetect β-glucanase, but not known related polypeptides using a standardWestern blot analysis. Examples of known related polypeptides areorthologs and proteins from the same species that are members of aprotein family.

Anti-β-glucanase antibodies can be produced using antigenic β-glucanaseepitope-bearing peptides and polypeptides. Antigenic epitope-bearingpeptides and polypeptides of the present invention contain a sequence ofat least nine, at least 12, at least 15, at least 18, at least 21, or atleast 24 to about 28 amino acids contained within SEQ ID NO:2. It isdesirable that the amino acid sequence of the epitope-bearing peptide isselected to provide substantial solubility in aqueous solvents (i.e.,the sequence includes relatively hydrophilic residues, while hydrophobicresidues are preferably avoided). Moreover, amino acid sequencescontaining proline residues may be also be desirable for antibodyproduction.

As an illustration, potential antigenic sites in β-glucanase can beidentified using the Jameson-Wolf method, Jameson and Wolf, CABIOS4:181, (1988), as implemented by the PROTEAN program (version 3.14) ofLASERGENE (DNASTAR; Madison, Wis.). Default parameters were used in thisanalysis.

The Jameson-Wolf method predicts potential antigenic determinants bycombining six major subroutines for protein structural prediction.Briefly, the Hopp-Woods method, Hopp et al., Proc. Nat'l Acad. Sci. USA78:3824 (1981), is first used to identify amino acid sequencesrepresenting areas of greatest local hydrophilicity (parameter: sevenresidues averaged). In the second step, Emini's method, Emini et al., J.Virology 55:836 (1985), is used to calculate surface probabilities(parameter: surface decision threshold (0.6)=1). Third, theKarplus-Schultz method, Karplus and Schultz, Naturwissenschaften 72:212(1985), is used to predict backbone chain flexibility (parameter:flexibility threshold (0.2)=1). In the fourth and fifth steps of theanalysis, secondary structure predictions are applied to the data usingthe methods of Chou-Fasman, Chou, “Prediction of Protein StructuralClasses from Amino Acid Composition,” in Prediction of Protein Structureand the Principles of Protein Conformation, Fasman (ed.), pages 549-586(Plenum Press 1990), and Garnier-Robson, Garnier et al., J. Mol. Biol.120:97 (1978) (Chou-Fasman parameters: conformation table=64 proteins; αregion threshold=103; β region threshold=105; Garnier-Robson parameters:α and β decision constants=0). In the sixth subroutine, flexibilityparameters and hydropathy/solvent accessibility factors are combined todetermine a surface contour value, designated as the “antigenic index.”Finally, a peak broadening function is applied to the antigenic index,which broadens major surface peaks by adding 20%, 40%, 60%, or 80% ofthe respective peak value to account for additional free energy derivedfrom the mobility of surface regions relative to interior regions. Thiscalculation is not applied, however, to any major peak that resides in ahelical region, since helical regions tend to be less flexible.

Polyclonal antibodies to recombinant β-glucanase protein or toβ-glucanase isolated from natural sources can be prepared using methodswell-known to those of skill in the art. Antibodies can also begenerated using a β-glucanase-glutathione transferase fusion protein,which is similar to a method described by Burrus and McMahon, Exp. Cell.Res. 220:363 (1995). General methods for producing polyclonal antibodiesare described, for example, by Green et al., “Production of PolyclonalAntisera,” in Immunochemical Protocols (Manson, ed.), pages 1-5 (HumanaPress 1992), and Williams et al., “Expression of foreign proteins in E.coli using plasmid vectors and purification of specific polyclonalantibodies,” in DNA Cloning 2: Expression Systems, 2nd Edition, Gloveret al. (eds.), page 15 (Oxford University Press 1995).

The immunogenicity of a β-glucanase polypeptide can be increased throughthe use of an adjuvant, such as alum (aluminum hydroxide) or Freund'scomplete or incomplete adjuvant. Polypeptides useful for immunizationalso include fusion polypeptides, such as fusions of β-glucanase or aportion thereof with an immunoglobulin polypeptide or with maltosebinding protein. The polypeptide immunogen may be a full-length moleculeor a portion thereof. If the polypeptide portion is “hapten-like,” suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

Although polyclonal antibodies are typically raised in animals such ashorse, cow, dog, chicken, rat, mouse, rabbit, goat, guinea pig, orsheep, an anti-β-glucanase antibody of the present invention may also bederived from a subhuman primate antibody. General techniques for raisingdiagnostically and therapeutically useful antibodies in baboons may befound, for example, in Goldenberg et al., International PatentPublication No. WO 91/11465, and in Losman et al., Int. J. Cancer 46:310(1990).

Alternatively, monoclonal anti-p-glucanase antibodies, e.g.,neutralizing monoclonal antibodies to neutralize β-glucanase activity,can be generated. Rodent monoclonal antibodies to specific antigens maybe obtained by methods known to those skilled in the art (see, forexample, Kohler et al., Nature 256:495 (1975), Coligan et al. (eds.),Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley &Sons 1991) [“Coligan”], Picksley et al., “Production of monoclonalantibodies against proteins expressed in E. coli,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (OxfordUniversity Press 1995)).

Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising a β-glucanase gene product, verifying thepresence of antibody production by removing a serum sample, removing thespleen to obtain B-lymphocytes, fusing the B-lymphocytes with myelomacells to produce hybridomas, cloning the hybridomas, selecting positiveclones which produce antibodies to the antigen, culturing the clonesthat produce antibodies to the antigen, and isolating the antibodiesfrom the hybridoma cultures.

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

For particular uses, it may be desirable to prepare fragments ofanti-β-glucanase antibodies. Such antibody fragments can be obtained,for example, by proteolytic hydrolysis of the antibody. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. As an illustration, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent to produce 3.5S Fab′monovalent fragments. Optionally, the cleavage reaction can be performedusing a blocking group for the sulfhydryl groups that result fromcleavage of disulfide linkages. As an alternative, an enzymatic cleavageusing pepsin produces two monovalent Fab fragments and an Fc fragmentdirectly. These methods are described, for example, by Goldenberg, U.S.Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys. 89:230(1960), Porter, Biochem. J. 73:119 (1959), Edelman et al., in Methods inEnzymology Vol. 1, page 422 (Academic Press 1967), and by Coligan atpages 2.8.1-2.8.10 and 2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described by Inbar etal., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde (see, for example,Sandhu, Crit. Rev. Biotech. 12:437 (1992)).

The Fv fragments may comprise VH and VL chains which are connected by apeptide linker. These single-chain antigen binding proteins (scFv) areprepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains which are connected by anoligonucleotide. The structural gene is inserted into an expressionvector which is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are described, for example, by Whitlow et al., Methods: ACompanion to Methods in Enzymology 2:97 (1991) (also see, Bird et al.,Science 242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack etal., Bio/Technology 11:1271 (1993), and Sandhu, supra).

As an illustration, a scFV can be obtained by exposing lymphocytes toβ-glucanase polypeptide in vitro, and selecting antibody displaylibraries in phage or similar vectors (for instance, through use ofimmobilized or labeled β-glucanase protein or peptide). Genes encodingpolypeptides having potential β-glucanase polypeptide binding domainscan be obtained by screening random peptide libraries displayed on phage(phage display) or on bacteria, such as E. coli. Nucleotide sequencesencoding the polypeptides can be obtained in a number of ways, such asthrough random mutagenesis and random polynucleotide synthesis. Theserandom peptide display libraries can be used to screen for peptideswhich interact with a known target which can be a protein orpolypeptide, such as a ligand or receptor, a biological or syntheticmacromolecule, or organic or inorganic substances. Techniques forcreating and screening such random peptide display libraries are knownin the art (Ladner et al., U.S. Pat. No. 5,223,409, Ladner et al., U.S.Pat. No. 4,946,778, Ladner et al., U.S. Pat. No. 5,403,484, Ladner etal., U.S. Pat. No. 5,571,698, and Kay et al., Phage Display of Peptidesand Proteins (Academic Press, Inc. 1996)) and random peptide displaylibraries and kits for screening such libraries are availablecommercially, for instance from CLONTECH Laboratories, Inc. (Palo Alto,Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.(Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway,N.J.). Random peptide display libraries can be screened using theβ-glucanase sequences disclosed herein to identify proteins which bindto β-glucanase.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106 (1991),Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press 1995), andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Polyclonal anti-idiotype antibodies can be prepared by immunizinganimals with anti-p-glucanase antibodies or antibody fragments, usingstandard techniques. See, for example, Green et al., “Production ofPolyclonal Antisera,” in Methods In Molecular Biology: ImmunochemicalProtocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, seeColigan at pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotypeantibodies can be prepared using anti-p-glucanase antibodies or antibodyfragments as immunogens with the techniques, described above.

Anti-idiotype β-glucanase antibodies, as well as β-glucanasepolypeptides, can be used to identify and to isolate β-glucanasesubstrates and inhibitors. For example, proteins and peptides of thepresent invention can be immobilized on a column and used to bindsubstrate and inhibitor proteins from biological samples that are runover the column (Hermanson et al. (eds.), Immobilized Affinity LigandTechniques, pages 195-202 (Academic Press 1992)). Radiolabeled oraffinity labeled β-glucanase polypeptides can also be used to identifyor to localize β-glucanase substrates and inhibitors in a biologicalsample (see, for example, Deutscher (ed.), Methods in Enzymol., vol.182, pages 721-37 (Academic Press 1990); Brunner et al., Ann. Rev.Biochem. 62:483 (1993); Fedan et al., Biochem. Pharmacol. 33:1167(1984)).

The present invention also provides DNA molecules, such as DNAconstructs containing a functional β-glucanase gene, in a kit.Alternatively, such a kit may include Pichia methanolica cells, such asdeficient in AUG1 and/or AUG2 promoter and vacuolar proteinase A and/orvacuolar proteinase B. Moreover, the kit may include instructions on howto insert a gene encoding a protein of interest into the DNA constructas well as instructions on how to tranform the provided Pichiamethanolica cells, and express, produce and recover the protein ofinterest.

The invention is further illustrated by the following nonlimitingexamples.

EXAMPLES Example 1 Identifiation of exo-1,3-β-glucanase

To clone the P. methanolica β-glucanase gene, a 45 kDa secreted proteinwas isolated from PMAD16 strain broth grown under fermentationconditions. N-terminal sequencing verified that the protein isolated wasfound to have 76.7% homology to the corresponding H. polymorphaexo-1,3-O-glucanase protein sequence and a 74.1% homology to thecorresponding S. occidentalis exo-1,3-O-glucanase protein sequencewithin a 30 amino acid overlap. Degenerate sense (ZC18,176; SEQ ID NO:7and ZCI8,177; SEQ ID.NO:8) and antisense (ZC16,562; SEQ ID NO:9 andZC16,567; SEQ ID NO:10 and ZCI8,180; SEQ ID NO:11 and ZC18,181; SEQ IDNO:12) PCR primers were designed from an alignment of the coding regionsof the exo-1,3-β-glucanase genes of H. polymorpha and S. occidentalis.The primers were then used to amplify P. methanolica genomic DNA. Anamplified sequence 1280 bp long was recovered and found to have 65.0%homology to the corresponding H. polymorpha exo-1,3-O-glucanase proteinsequence.

A P. methanolica genomic library was constructed in the vector pRS426(Christianson et al., Gene 110:119-122, 1992), a shuttle vectorcomprising 2μ and S. cerevisiae URA3 sequences, allowing it to bepropagated in S. cerevisiae. Genomic DNA was prepared from strainCBS6515 according to standard procedures. Briefly, cells were culturedovernight in rich media, spheroplasted with zymolyase, and lysed withSDS. DNA was precipitated from the lysate with ethanol and extractedwith a phenol/chloroform mixture, then precipitated with ammoniumacetate and ethanol. Gel electrophoresis of the DNA preparation showedthe presence of intact, high molecular weight DNA and appreciablequantities of RNA. The DNA was partially digested with Sau 3A byincubating the DNA in the presence of a dilution series of the enzyme.Samples of the digests were analyzed by electrophoresis to determine thesize distribution of fragments. DNA migrating between 4 and 12 kb wascut from the gel and extracted from the gel slice. The size-fractionatedDNA was then ligated to pRS426 that had been digested with Bam HI andtreated with alkaline phosphatase. Aliquots of the reaction mixture wereelectroporated into E. coli MC 1061 cells using an electroporator (GenePulserm; BioRad Laboratories, Hercules, Calif.) as recommended by themanufacturer.

The library was screened by PCR using sense and antisense primersdesigned from the sequenced region of the P. methanolicaexo-1,3-O-glucanase gene fragment. The PCR reaction mixture wasincubated for one minute at 94° C.: followed by 34 cycles of 94° C., oneminute, 52° C., one minute, 72° C., eleven minutes. Starting with 43library pools, positive pools were identified and broken down toindividual colonies. A single colony with a pRS426 plasmid containingthe P. methanolica exo-1,3-O-glucanase gene as its insert was isolated.The orientation of the exo-1,3-glucanase gene and the length of the 5′and 3′ flanking sequences in the insert were deduced by DNA sequencing(SEQ ID NO:1). This gene was designated exo-1,3-β-glucanase.

Example 2 Construction and Characterization of ZACRP3 Untagged YeastExpression Vectors Utilizing a Heterologous S. cerevisiae Leader and anEndogenous P. methanolica Leader

Expression of zacrp3 (Piddington et al., U.S. Pat. No. 6,521,233) inPichia methanolica utilizes the expression system as described inRaymond, U.S. Pat. No. 5,888,768; Raymond, U.S. Pat. No. 5,955,349; andRaymond, U.S. Pat. No. 6,001,597. An expression plasmid containing allor part of a polynucleotide encoding zacrp3 is constructed viahomologous recombination (Raymond et al., U.S. Pat. No. 5,854,039). Anexpression vector was built from pVRM51 to express untagged zacrp3polypeptides. PVRM51 is a derivative of the pCZR204 expression vector;it differs from pCZR204 by one amino acid (D83->Y83) within the alphafactor prepro (aFpp) sequence to enhance Kex2p cleavage. The pVRM51vector contains the AUG1 promoter, followed by the aFpp (D83->Y83)leader sequence and an amino-terminal peptide tag (Glu-Glu), followed bya blunt-ended Sma I restriction site, a carboxy-terminal peptide tag(Glu-Glu), a translational STOP codon, followed by the AUG1 terminator,the ADE2 selectable marker, and finally the AUG1 3′ untranslated region.Also included in this vector are the URA3 and CEN-ARS sequences requiredfor selection and replication in S. cerevisiae, and the AmpR and colE1ori sequences required for selection and replication in E. coli. Asecond expression vector was built from zCZR204 to express untaggedzacrp3 polypeptides. The zCZR204 expression vector is as describedabove, the only difference is that this expression plasmid has theβ-glucanase leader inserted where the aFpp leader usually is. The zacrp3sequence inserted into these vectors begins at residue 23 (Gln) of thezacrp3 amino acid sequence. The nucleotide sequence of zacrp3 is shownin SEQ ID NO:13 and the polypeptide sequence of zacrp3 is shown in SEQID NO:14.

For each construct specific recombination primers were designed. For theaFppD->Y::zacrp3 construct, these primers are ZG37,475 (SEQ ID NO:15)and ZG37,474 (SEQ ID NO:16). For the β-glucanase::zacrp3 construct, theβ-glucanase leader was amplified using primers ZG39,207 (SEQ ID NO:17)and ZG39,209 (SEQ ID NO:18), while zacrp3 was amplified using primersZG39,208 (SEQ ID NO:19) and ZG37,474 (SEQ ID NO:16). The resulting PCRfragments were homologously recombined into the yeast expression vectorsdescribed above. For the aFppD->Y::zacrp3 construct, the N-terminalprimer (ZG37,475) (SEQ ID NO:15) spans 39 base pairs of the alpha factorprepro (aFpp) coding sequence on one end, followed by 26 base pairs ofthe amino-terminus coding sequence of mature zacrp3 sequence on theother. The C-termiinal primer (ZG37,474) (SEQ ID NO:16) spans about 28base pairs of carboxy terminus coding sequence of zacrp3 on one end with40 base pairs of AUG1 terminator sequence.

For the β-glucanase::zacrp3 construct, the N-terminal β-glucanase primer(ZG39,207) (SEQ ID NO:17) spans 40 base pairs of AUGIp sequence,followed by 27 base pairs of β-glucanase leader sequence. The C-terminalprimer (ZG39,209) (SEQ ID NO:18) that amplifies β-glucanase contains 30base pairs of carboxy terminus coding sequence of β-glucanase followedby 33 base pairs of the amino-terminus coding sequence of the Glu-Glutag. The N-terminal zacrp3 primer (ZG39,208) (SEQ ID NO:19) spans 39base pairs of β-glucanase sequence, followed by 26 base pairs of themature zacrp3 sequence. The C-terminal primer (ZG37,474) (SEQ ID NO:16)that amplifies zacrp3 spans about 28 base pairs of carboxy terminuscoding sequence of zacrp3 on one end with 40 base pairs of AUG1terminator sequence.

Construction of the Untagged zacrp3 Plasmid Utilizing the aFpp Leader

An untagged zacrp3 plasmid was made by homologously recombining 100 ngof the SmaI digested pVRM51 acceptor vector and 1 μg of PCR amplifiedzacrp3 cDNA donor fragment, in S. cerevisiae SF838-9Da.

The zacrp3 PCR fragment was synthesized by a PCR reaction. To a finalreaction volume of 100 μl was added 100 pmol each of primers, ZG37,474(SEQ ID NO:16) and ZG37,475 (SEQ ID NO:15), 10 μl of 10× PCR buffer(Boehringer Mannheim), 1 μl Pwo Polymerase (Boehringer Mannheim), 10 μlof 0.25 mM nucleotide triphosphate mix (Perkin Elmer) and dH₂O. The PCRreaction was run 1 cycle at 2 minutes at 94° C., followed by 25. cyclesof 30 seconds at 94° C., 1 minute at 50° C. and 1 minute at 72° C.,followed by a 7 minute extension at 72° C., and concluded with anovernight hold at 4° C. The resulting 754 bp double stranded, zacrp3fragment is disclosed in SEQ ID NO:20.

Construction of the Untagged zacrp3 Plasmid Utilizing the β-GlucanaseLeader

An untagged zacrp3 plasmid was made by homologously recombining 100 ngof the SmaI digested pCZR204 acceptor vector and 1 μg each of PCRamplified β-glucanase leader donor fragment and 1>g zacrp3 cDNA donorfragment, in S. cerevisiae SF838-9Da. The zacrp3 PCR fragments weresynthesized by first amplifying the two fragments containing theβ-glucanase leader and zacrp3, respectively, in separate reactions.

The β-glucanase leader was amplified in a PCR reaction as follows: to afinal reaction volume of 100 μl was added 100 μmol each of primers,ZG39,207 (SEQ ID NO:17) and ZG39,209 (SEQ ID NO:18), 10 μl of 10× PCRbuffer (Boehringer Mannheim), 1 μl Pwo Polymerase (Boehringer Mannheim),10 μl of 0.25 mM nucleotide triphosphate mix (Perkin Elmer) and dH₂O.The PCR reaction was run 1 cycle at 2 minutes at 94° C., followed by 25cycles of 30 seconds at 94° C., 1 minute at 50° C. and 30 seconds at 72°C., followed by a 7 minute extension at 72° C., and concluded with anovernight hold at 4° C. The resulting 157 bp double stranded,β-glucanase leader fragment is disclosed in SEQ ID NO:21.

Zacrp3 was amplified in an additional PCR reaction as follows: to afinal reaction volume of 100 μl was added 100 μmol each of primers,ZG39,208 (SEQ ID NO:19) and ZG37,474 (SEQ ID NO:16), 10 μl of 10× PCRbuffer (Boehringer Mannheim), 1 μl Pwo Polymerase (Boehringer Mannheim),10 μl of 0.25 mM nucleotide triphosphate mix (Perkin Elmer) and dH₂O.The PCR reaction was run 1 cycle at 2 minutes at 94° C., followed by 25cycles of 30 seconds at 94° C., 1 minute at 50° C. and 30 seconds at 72°C., followed by a 7 minute extension at 72° C., and concluded with anovernight hold at 4° C. The resulting 754 bp fragment is doublestranded, and the zacrp3 PCR fragment is disclosed in SEQ ID NO:22.

One hundred microliters of competent yeast cells (S. cerevisiae strainSF838-9Da) was independently combined with the various DNA mixtures fromabove and transferred to a 0.2 cm electroporation cuvette. The yeast/DNAmixtures were electropulsed at 0.75 kV (5 kV/cm), infinite Ω, 25 μF. Theyeast/DNA mixtures were then added to 1 ml of 1.2 M sorbitol andincubated at 30° C. for 1 hour. The yeast was then plated in two 500 μlaliquots onto two URA DS plates and incubated at 30° C.

After about 48 hours the Ura+yeast transformants from a single platewere resuspended in 1 ml H₂O and spun briefly to pellet the yeast cells.The cell pellet was resuspended in 300 μl of Qiagen P1 lysis buffer andtransferred to a fresh tube that contained 100-200 μl acid-washed glassbeads (Sigma). Samples were vortexed for 1 minute intervals two or threetimes to lyse cells. Samples were allowed to settle, and 250 μl lysatewas transferred to a fresh tube and the remainder of the Qiagen SpinMiniprep Kit was carried out following manufacterer's instructions.

Transformation of electrocompetent E. coli DH10B cells (Invitrogen) wasdone with 2 μl yeast DNA prep and 40 μl of DH10B cells. The cells wereelectropulsed in 0.1 cm cuvettes at 2.0 kV, 25 μF and 100 Ω. Followingelectroporation, 250 μl SOC (2% Bacto Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mMMgSO₄, 20 mM glucose) was plated in one aliquot on an LB AMP plate (LBbroth (Lennox), 1.8% Bacto Agar (Difco), 100 mg/L Ampicillin). Plateswere incubated at 37° C. overnight.

Individual clones harboring the correct expression construct foruntagged zacrp3 were identified by restriction digest to verify thepresence of the zacrp3 insert and to confirm that the various DNAsequences had been joined correctly to one another. The inserts ofpositive clones were subjected to sequence analysis. The afpp D->Yleader::zacrp3 plasmid was designated pSDH147 and the β-glucanaseleader::zacrp3 plasmid was designated pSDH149. Larger scale plasmid DNAwas isolated for both plasmids using the Qiagen Maxi kit (Qiagen)according to manufacturer's instruction and the DNA was digested withNot I to liberate the Pichia-zacrp3 expression cassette from the vectorbackbone. The Not I-restriction digested DNA fragment was thentransformed into the Pichia methanolica expression hosts, PMAD16 andPMAD18. This was done by mixing 100 μl of prepared competent PMAD16 orPMAD18 cells with 10 μg of Not I restriction digested pSDH147 orpSDH149, in separate transformations, and transferred to a 0.2 cmelectroporation cuvette. The yeast/DNA mixture was electropulsed at 0.75kV, 25 μF, infinite Ω. To the cuvette was added 800 μl of 1.2M Sorbitoland 400 μl aliquots were plated onto two ADE DS (0.056%-Ade-Trp-Thrpowder, 0.67% yeast nitrogen base without amino acids, 2% D-glucose,0.5% 200× tryptophan, threonine solution, and 18.22% D-sorbitol) platesfor selection and incubated at 30° C.

Zacrp3 Expression in P. methanolica Hosts PMAD16 and PMAD18-CloneSelection and Characterization

One hundred clones of each strain/plasmid (for 400 clones total) wereisolated. Of these, only 10 of each were screened via Western blot forhigh-level zacrp3 expression. All 40 clones were grown in the followingmanner: 25 ml cultures of each were inoculated using one colony of eachstrain in BMY.1 pH6.0 media (Per liter: 13.4 g Yeast Nitrogen Basewithout amino acids (Becton Dickinson), 10.0 g Yeast Extract (Difco),10.0 g tryptone (Difco), 10.0 g casamino acids (Difco), 6.7 g K₂HPO₄ (EMScience), 4.2 g citric acid (EM Science), and water)+2% glucose. BMY.1media was supplemented with 10 mis per liter of media with FXIII vitaminsolution (0.05 g/L biotin, 0.8 g/L thiamine hydrochloride, 0.8 g/Lpyroxidine HCL, 15.0 g/L inositol, 15.0 g/L calcium pantothenate, 0.6g/L niacinamide, 0.1 g/L folic acid, 0.2 g/L riboflavin, 1.0 g/L cholinechloride). Cultures were grown in 125 ml baffled flasks on a platformshaker set to 250 rpm at 30° C. overnight.

The following day, 1 ml of each overnight inoculum culture was dilutedinto 24 mls of fresh BMY.1 media supplemented with FXIII vitamins asabove, +1% Methanol to induce the AUG1 promoter (no glucose was added).Cultures were grown in 125 ml baffled flasks on a platform shaker set to250 rpm at 30° C. for 24 hours. After 24 hours of growth and induction,the cultures were harvested at 5000 rpm for 10 minutes in a Beckmancentrifuge (JA-20 rotor) to pellet the cells. Three hundred μl of zacrp3containing supernatant was mixed with 100 μl of NuPAGE 4× Sample Buffer(Invitrogen). Each 400 μl sample was split into two 200 μl samples: oneset of samples was treated with 2% β-mercaptoethanol (Sigma) andrepresents a reduced sample, while the other set represents thenon-reduced sample.

An SDS-PAGE analysis was carried out as described below. All reducedsamples were heated for 10 min at 100° C., while all non-reduced sampleswere heated for 10 min at 65° C. Fifteen μL of each sample was appliedfor electrophoresis on a polyacrylamide gel. Protein separation wasperformed by electrophoresis in a 4-12% gradient NuPAGE polyacrylamideresolving gel (Invitrogen) under denaturing conditions (SDS-PAGE) using1×MES running buffer (Invitrogen). The voltage of 130V was appliedthroughout the entire run. Subsequently, electrotransference was carriedout to a 0.2 μn nitrocellulose membrane (Invitrogen) for 1 h at 400 mA(constant current). The blots were then incubated for 30 minutes withagitation at 40 rpm in a blocking solution [Western A+10% non-fat drymilk (NFDM)(Carnation)] in order to block the protein-free areas of themembrane at 25° C.

As the first antibody, an anti-zacrp3 affinity purified antibody, E1834,developed in the rabbit (in-house) was used in a dilution of 1:10,000 inWestern A+2.5% NFDM. Incubation was 2 hours at 25° C. Subsequently two 5min. washings were performed at moderate agitation with Western B,followed by one 5 minute was at moderate agitation with Western A. Asthe second antibody a rabbit anti-IgG developed in the goat (Amersham)was used in a dilution of 1:2000 in Western A+2.5% NFDM. Blots wereincubated for 1 hour at room temperature and washed three times for 5min with moderate agitation with Western B, followed by a brief rinse indH₂O. Two mls of both Enhanced Chemiluminescent substrates (Amersham)were mixed together at a 1:1 ratio, and the blots were incubated in thissolution for 5 seconds prior to development. The exposed blots were thendeveloped using timed exposure to X-ray film (Kodak) and the film wassubsequently developed to visualize data.

The electrophoretic analysis on the polyacrylamide gel of the culturemedium from P. methanolica clones representing pSDH149 (β-glucanaseleader) and pSDH147 (S. cerevisiae alpha factor pre-pro sequence) showedthat in the culture medium from both host strains a band ofapproximately 28 kDa (under reduced conditions) appears corresponding tozacrp3, while in the non-induced cell culture medium, there was no band.Roughly ninety percent of the recombinant clones that were analyzed forthe integrated heterologous gene expression produced and secretedrecombinant zacrp3. The resulting zacrp3 plasmid-containing yeaststrains show the endogenous P. methanolica β-glucanase leader constructpSDH149 secretes equivalent levels of zacrp3 compared to theheterologous S. cerevisiae aFpp leader pSDH147 in the PMAD16 host strainbackground. Interestingly, plasmid-containing yeast strains show theendogenous P. methanolica β-glucanase leader construct pSDH149 secretesapproximately 2-3 fold higher levels of zacrp3 compared to theheterologous S. cerevisiae aFpp leader pSDH147 in the PMAD18 host strainbackground. One isolet of each aFpp::zacrp3 strain was picked forsubsequent use; the resulting clones were designatedPMAD16::pSDH147.4.2, PMAD18::pSDH147.4.8, respectively. Two isolets ofeach β-glucanase::zacrp3 strain was picked for subsequent use; theresulting clones were designated PMAD16::pSDH149.4.4,PMAD16::pSDH149.4.9, PMAD18::pSDH149.4.5, and PMAD18::pSDH149.4.8,respectively.

Example 3 Construction and Characterization of Zsig37 Untagged YeastExpression Vectors Utilizing a Heterologous S. cerevisiae Leader and anEndogenous P. methanolica Leader

Expression of zsig37 in Pichia methanolica utilizes the expressionsystem as described in Raymond, U.S. Pat. No. 5,888,768; Raymond, U.S.Pat. No. 5,955,349; and Raymond, U.S. Pat. No. 6,001,597. An expressionplasmid containing all or part of a polynucleotide encoding zsig37 isconstructed via homologous recombination (Raymond et al., U.S. Pat. No.5,854,039). Zsig37 was recombined into the vector pCZR204. Oligos usedto amplify zsig37 introduced a single amino acid mutation (D83->Y83)within the alpha factor prepro (aFpp) sequence to enhance Kex2pcleavage. This mutation was then introduced into the vector pCZR204 whenrecombination occurred. The pCZR204 vector contains the AUG1 promoter,followed by the afpp leader sequence and an amino-terminal peptide tag(Glu-Glu), followed by a blunt-ended Sma I restriction site, acarboxy-terminal peptide tag (Glu-Glu), a translational STOP codon,followed by the AUG1 terminator, the ADE2 selectable marker, and finallythe AUG1 3′ untranslated region. Also included in this vector are theURA3 and CEN-ARS sequences required for selection and replication in S.cerevisiae, and the AmpR and colE1 ori sequences required for selectionand replication in E. coli. A second expression vector was built fromzCZR204 to express untagged zsig37 polypeptides. The zCZR204 expressionvector is as described above, the only difference is that thisexpression plasmid has the β-glucanase leader inserted where the aFppleader usually is. The zsig37 sequence inserted into these vectorsbegins at residue 86 (Arg) of the zsig37 amino acid sequence. Thefull-length nucleotide sequence of zsig37 is shown in SEQ ID NO:27 andthe full-length polypeptide sequence of zsig37 is shown in SEQ ID NO:28(See U.S. Pat. Nos. 6,265,544, 6,566,499, 6,518,403, 6,448,221, and6,544,946).

For each construct specific recombination primers were designed. For theaFppD83->Y83::zsig37 construct, these primers are ZG42,210 (SEQ IDNO:29) and ZG42,206 (SEQ ID NO:30). For the β-glucanase::zsig37construct, the β-glucanase leader was amplified using primers ZG42,209(SEQ ID NO:31) and ZG42,211 (SEQ ID NO:32), while zsig37 was amplifiedusing primers ZG42,273 (SEQ ID NO:33) and ZG42,206 (SEQ ID NO:30). Theresulting PCR fragments were homologously recombined into the yeastexpression vector described above. For the aFppD83->Y83::zsig37construct, the N-terminal primer (ZG42,210) (SEQ ID NO:29) spans 39 basepairs of the alpha factor prepro (aFpp) coding sequence on one end, andintroduces the D83->Y83 mutation in the aFpp sequence, followed by 25base pairs of the amino-terminus coding sequence of mature zsig37sequence on the other. The C-terminal primer (ZG42,206) (SEQ ID NO:30)spans about 21 base pairs of carboxy terminus coding sequence of zsig37on one end with 40 base pairs of AUG1 terminator sequence.

For the β-glucanase::zsig37 construct, the N-terminal β-glucanase primer(ZG42,209) (SEQ ID NO:31) spans 40 base pairs of AUGIp sequence,followed by 27 base pairs of β-glucanase leader sequence. The C-terminalprimer (ZG42,211) (SEQ ID NO:32) that amplifies α-glucanase contains 39base pairs of carboxy terminus coding sequence of β-glucanase followedby 25 base pairs of the amino-terminus coding sequence of the maturezsig37 sequence. The N-terminal zsig37 primer (ZG42,273) (SEQ ID NO:33)spans 39 base pairs of β-glucanase sequence, followed by 25 base pairsof the mature zsig37 sequence. The C-terminal primer (ZG42,206) (SEQ IDNO:30) that amplifies zsig37 spans about 21 base pairs of carboxyterminus coding sequence of zsig37 on one end with 40 base pairs of AUG1terminator sequence.

Construction of the Untagged zsig37 Plasmid Utilizing the aFppD->YLeader

An untagged zsig37 plasmid was made by homologously recombining 100 ngof the SmaI digested pCZR204 acceptor vector and 1 μg of PCR amplifiedzsig37 cDNA donor fragment, in S. cerevisiae SF838-9Da.

The zsig37 PCR fragment was synthesized by a PCR reaction. To a finalreaction volume of 100 μl was added 100 μmol each of primers, ZG42,210(SEQ ID NO:29) and ZG42,206 (SEQ ID NO:30), 10 μl of 10× PCR buffer(Boehringer Mannheim), 1 μl Pwo Polymerase (Boehringer Mannheim), 10 μlof 0.25 mM nucleotide triphosphate mix (Perkin Elmer) and dH₂O. The PCRreaction was run 1 cycle at 2 minutes at 94° C., followed by 30 cyclesof 30 seconds at 94° C., 1 minute at 50° C. and 1 minute at 72° C.,followed by a 7 minute extension at 72° C., and concluded with anovernight hold at 4° C. The resulting 846 bp double stranded, zsig37fragment is disclosed in SEQ ID NO:34. The aFpp:zsig37 full-lengthnucleotide (pSDH156) is shown in SEQ ID NO:35, with its correspondingencoded protein shown in SEQ ID NO:36.

Construction of the Untagged zsig37 Plasmid Utilizing the α-GlucanaseLeader

An untagged zsig37 plasmid was made by homologously recombining 100 ngof the SmaI digested pCZR204 acceptor vector and 1 μg each of PCRamplified β-glucanase leader donor fragment and 1 μg zsig37 cDNA donorfragment, in S. cerevisiae SF838-9Da. The zsig37 PCR fragments weresynthesized by first amplifying the two fragments containing theβ-glucanase leader and zsig37, respectively, in separate reactions.

The β-glucanase leader was amplified in a PCR reaction as follows: to afinal reaction volume of 100 μl was added 100 μmol each of primers,ZG42,209 (SEQ ID NO:31) and ZG42,211 (SEQ ID NO:32), 10 μl of 10×PCRbuffer (Boehringer Mannheim), 1 μl Pwo Polymerase (Boehringer Mannheim),10 μl of 0.25 mM nucleotide triphosphate mix (Perkin Elmer) and dH₂O.The PCR reaction was run 1 cycle at 2 minutes at 94° C., followed by 30cycles of 30 seconds at 94° C., 1 minute at 50° C. and 1 minute at 72°C., followed by a 7 minute extension at 72° C., and concluded with anovernight hold at 4° C. The resulting 148 bp double stranded,β-glucanase leader fragment is disclosed in SEQ ID NO:37.

Zsig37 was amplified in an additional PCR reaction as follows: to afinal reaction volume of 100 μl was added 100 μmol each of primers,ZG42,273 (SEQ ID NO:33) and ZG42,206 (SEQ ID NO:30), 10 μl of 10×PCRbuffer (Boehringer Mannheim), 1 μl Pwo Polymerase (Boehringer Mannheim),10 μl of 0.25 mM nucleotide triphosphate mix (Perkin Elmer) and dH₂O.The PCR reaction was run 1 cycle at 2 minutes at 94° C., followed by 30cycles of 30 seconds at 94° C., 1 minute at 50° C. and 1 minute at 72°C., followed by a 7 minute extension at 72° C., and concluded with anovernight hold at 4° C. The resulting 846 bp fragment is doublestranded, and the zsig37 PCR fragment is disclosed in SEQ ID NO:38.

One hundred microliters of competent yeast cells (S. cerevisiae strainSF838-9Da) was independently combined with the various DNA mixtures fromabove and transferred to a 0.2 cm electroporation cuvette. The yeast/DNAmixtures were electropulsed at 0.75 kV (5 kV/cm), infinite Ω, 25 μF. Theyeast/DNA mixtures were then added to 1 ml of 1.2 M sorbitol andincubated at 30° C. for 1 hour. The yeast was then plated in two 500 μlaliquots onto two URA DS plates and incubated at 30° C.

After about 48 hours the Ura+yeast transformants from a single platewere resuspended in 1 ml H₂O and spun briefly to pellet the yeast cells.The cell pellet was resuspended in 300 μl of Qiagen P1 lysis buffer andtransferred to a fresh tube that contained 100-200 μl acid-washed glassbeads (Sigma). Samples were vortexed for 1 minute intervals two or threetimes to lyse cells. Samples were allowed to settle, and 250 μl lysatewas transferred to a fresh tube and the remainder of the Qiagen SpinMiniprep Kit was carried out following manufacterer's instructions.

Transformation of electrocompetent E. coli DH10B cells (Invitrogen) wasdone with 2 μl yeast DNA prep and 40 μl of DH10B cells. The cells wereelectropulsed in 0.1 cm cuvettes at 2.0 kV, 25 μF and 100 Ω. Followingelectroporation, 250 μl SOC (2% Bacto Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl (J. T. Baker), 2.5 mM KCl(Mallinkrodt), 10 mM MgCl₂ (Mallinkrodt), 10 mM MgSO₄ (J. T. Baker), 20mM glucose (Difco) and water) was plated in one aliquot on an LB AMPplate (LB broth (Lennox), 1.8% Bacto Agar (Difco), 100 mg/L Ampicillin(Sigma)). Plates were incubated at 37° C. overnight.

Individual clones harboring the correct expression construct foruntagged zsig37 were identified by restriction digest to verify thepresence of the zsig37 insert and to confirm that the various DNAsequences had been joined correctly to one another. The inserts ofpositive clones were subjected to sequence analysis. The aFpp D83->Y83leader::zsig37 plasmid was designated pSDH156 and the β-glucanaseleader::zsig37 plasmid was designated pSDH160. Larger scale plasmid DNAwas isolated for both plasmids using the Qiagen Maxi kit (Qiagen)according to manufacturer's instruction and the DNA was digested withNot I to liberate the Pichia-zsig37 expression cassette from the vectorbackbone. The Not I-restriction digested DNA fragment was thentransformed into the Pichia methanolica expression hosts, PMAD16 andPMAD18. This was done by mixing 100 μl of prepared competent PMAD16 orPMAD18 cells with 1.0 μg and 2.5 μg of Not I restriction digestedpSDH156 or pSDH160, in separate transformations, and transferred to a0.2 cm electroporation cuvette. The yeast/DNA mixture was electropulsedat 0.75 kV, 25 μF, infinite Q. To the cuvette was added 800 μl of 1.2MSorbitol. Transformants were outgrown in test tubes at 30° C. for 2hours prior to plating on selection plates. Four hundred μl aliquotswere plated onto two ADE DS (0.056%-Ade-Trp-Thr powder (TCI America,Alfa Aesar, and Calbiochem), 0.67% yeast nitrogen base without aminoacids (Becton Dickinson), 2% D-glucose (Difco), 0.5% 200× tryptophan,threonine solution (ICN and Alfa Aesar), and 18.22% D-sorbitol) platesfor selection and incubated at 30° C. The β-glucanase::zsig37full-length nucleotide sequence (pSDH160) is shown in SEQ ID NO:39, withits corresponding encoded protein shown in SEQ ID NO:40.

Zsig37 Expression in P. methanolica Hosts PMAD16 and PMAD18-CloneSelection and Characterization

Two hundred fifty clones of PMAD16::pSDH156 and 300 clones ofPMAD18::pSDH156 were isolated. In addition, 55 clones of PMAD16::pSDH160and 68 clones of PMAD18::pSDH160 were isolated. All clones were screenedvia colony blot analysis for high-level zsig37 expression. Clones werescreened by colony blot as follows: each transformant was patched to twofresh 1% Methanol plates (Per liter: 6.8 g Yeast Nitrogen Base withoutamino acids (Becton Dickinson), 0.6 g-ade-trp-thr powder (TCI America,Alfa Aesar, Calbiochem), 18.0 g Bacto agar (Difco), 5 mls 200×Tryptophan/threonine solution (Alfa Aesar and ICN), 10 mls Methanol (J.T. Baker), 2 mis saturated biotin (ICN) and water). Each plate wasoverlayed with a nitrocellulose filter (Schleicher & Schuell) andincubated at 30° C. for 3 days. Nitrocellulose filters were thenremoved. One set of filters was denatured and reduced under thefollowing conditions: filters were placed in a hybridization tube and 25mls of 25 mM Tris (Millipore), 25 mM Glycine (J. T. Baker), 5 mM β-ME(Sigma) pH9.0 was added to each tube. Filters were incubated at 65° C.for 10 minutes. Post-denaturation/reduction, filters were removed andplaced directly in Western block solution (50 mM Tris (Millipore) pH7.4,5 mM EDTA (J. T. Baker) pH8.0, 0.05% Igepal CA-630 (Sigma), 150 mM NaCl(J. T. Baker), 2.5% Gelatin (Mallinkrodt), water and 10% nonfat dry milk(NFDM)(Carnation)). The other identical set of filters represents anon-denatured, non-reduced set of filters. These filters were removedfrom the plates and placed directly into Western block solution. Allfilters were incubated in block solution for 30 minutes at 25° C.

Filters were then incubated in Western A (50 mM Tris (Millipore) pH7.4,5 mM EDTA (J. T. Baker) pH8.0, 0.05% Igepal CA-630 (Sigma), 150 mM NaCl(J. T. Baker), 2.5% Gelatin (Mallinkrodt), water)+2.5% NFDM (Carnation)containing 0.2 μg/ml zsig37 primary antibody E1489 for 1-2 hours at 25°C. Blots were then washed 3 times for 7 minutes each at 25° C. inWestern B (1M NaCl (J. T. Baker), 50 mM Tris (Millipore) pH7.4, 5 mMEDTA (J. T. Baker), 0.05% Igepal (Sigma), 0.25% gelatin (Mallinkrodt),and water) followed by one wash in Western A for 7 minutes at 25° C.Filters were then incubated in Western A+2.5% NFDM containing a 1:5000dilution of donkey anti rabbit secondary antibody (Life Technologies)for 1 hour at 25° C. Blots were then washed 4 times for 7 minutes eachat 25° C. in Western B (1M NaCl (J. T. Baker), 50 mM Tris (Millipore)pH7.4, 5 mM EDTA (J. T. Baker), 0.05% Igepal (Sigma), 0.25% gelatin(Mallinkrodt), and water) at 25° C. All blots were then briefly rinsedwith deionized water before being developed with Lumi-Light Plus ECLsubstrate (Roche). Two mls of both Lumi-Light substrates were mixedtogether at a 1:1 ratio, and the blots were incubated in this solutionfor 5 seconds prior to development. The exposed blots were thendeveloped using timed exposure to X-ray film (Kodak) and the film wassubsequently developed to visualize data.

Ten clones of PMAD16::pSDH156, 12 clones of PMAD18::pSDH156, 6 clones ofPMAD16::pSDH160 and 6 clones of PMAD18::pSDH160 were picked forfollow-up western analysis. All clones were grown in the followingmanner: 5 ml cultures of each were inoculated using one colony of eachstrain in YEPD media (Per liter: 20.0 g D-Glucose (J. T. Baker), 20.0 gBacto Peptone (Difco), 10.0 g Yeast Extract (Difco), 0.04 g adenine(Alfa Aesar), 0.06 g L-Leucine (TCI America) and water). Cultures weregrown in test tubes and placed on a roller drum at 30° C. overnight. Thefollowing day, 0.5 ml of each overnight inoculum culture was dilutedinto 24.5 mls of BMY.1 media (Per liter: 13.4 g Yeast Nitrogen Basewithout amino acids (Becton Dickinson), 10.0 g Yeast Extract (Difco),10.0 g tryptone (Difco), 10.0 g casamino acids (Difco), 6.7 g K₂HPO₄ (EMScience), 4.2 g citric acid (EM Science), and water) supplemented with10 mls per liter of media with FXIII vitamin solution (0.05 g/L biotin,0.8 g/L thiamine hydrochloride, 0.8 g/L pyroxidine HCL, 15.0 g/Linositol, 15.0 g/L calcium pantothenate, 0.6 g/L niacinamide, 0.1 g/Lfolic acid, 0.2 g/L riboflavin, 1.0 g/L choline chloride) and 10 mls perliter of Methanol (J. T. Baker) for a 1% Methanol final concentration.Cultures were grown in 125 ml baffled flasks on a platform shaker set to250 rpm at 30° C. for 48 hours. After 24 hours, a sample was taken forwestern analysis, and a 1% Methanol dose was added to each culture.

After 48 hours of growth and induction, the cultures were harvested at10,000 rpm for 10 minutes in a Beckman centrifuge (JA-20 rotor) topellet the cells. Two hundred fifty μl of zsig37 containing supernatantwas mixed with 250 μl of 2× Laemmli Sample Buffer (125 mM Tris(Millipore), 20% glycerol (EM Science), 4% SDS (ICN), 0.01% Bromophenolblue (EM Science) and water). Each 500 μl sample was split into two 250μl samples: one set of samples was treated with 2% β-mercaptoethanol(Sigma) and represents a reduced sample, while the other set representsthe non-reduced sample.

An SDS-PAGE analysis was carried out as described below. All reducedsamples were heated for 10 min at 65° C., while all non-reduced sampleswere not heated. Fifteen μL of each sample was applied forelectrophoresis on a polyacrylamide gel. Protein separation wasperformed by electrophoresis in a 4-12% gradient Tris-Gly polyacrylamideresolving gel (Invitrogen) under denaturing conditions (SDS-PAGE) using1× Glycine running buffer (Invitrogen). The voltage of 80V was appliedfor the first 30 minutes, then the voltage was raised to 130V for theduration of the run. Subsequently, electrotransference was carried outto a 0.2 μm nitrocellulose membrane (Invitrogen) for 2 h at 200 mA(constant current). The blots were then developed as above.

The electrophoretic analysis on the polyacrylamide gel of the culturemedium from P. methanolica clones representing pSDH156 (S. cerevisiaealpha factor D->Y pre-pro sequence) and pSDH160 (β-glucanase leader)showed that in the culture medium from both host strains a milieuappears corresponding to various zsig37 forms, while in the non-inducedcell culture medium, there was no band. Roughly ninety percent of therecombinant clones that were analyzed for the integrated heterologousgene expression produced and secreted recombinant zsig37. The resultingzsig37 plasmid-containing yeast strains show the heterologous S.cerevisiae aFpp construct pSDH156 secretes equivalent levels of zsig37compared to the endogenous P. methanolica β-glucanase leader pSDH160 inthe PMAD16 host strain background. Interestingly, plasmid-containingyeast strains show the endogenous P. methanolica β-glucanase leaderconstruct pSDH160 secretes approximately 2-3 fold higher levels ofzsig37 in PMAD16 compared to the PMAD18 host strain background. Everyisolet of each aFpp::zsig37 strain was picked for subsequent use; theresulting clones were designated PMAD16::pSDH156 isolets #40, 56, 58,84, 92, 149, 167, 169, 230, 231, and PMAD18::pSDH156 isolets #23, 29,35, 144, 149, 161, 191, 202, 206, 217, 224, 269, respectively. Inaddition, every isolet of each β-glucanase::zsig37 strain was picked forsubsequent use; the resulting clones were designated PMAD16::pSDH160isolets #1, 2, 26, 30, 44, and PMAD18::pSDH160 isolets #1, 10, 21, 43,48, 62, respectively.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (e.g., GenBank aminoacid and nucleotide sequence submissions) cited herein are incorporatedby reference. The foregoing detailed description and examples have beengiven for clarity of understanding only. No unnecessary limitations areto be understood therefrom. The invention is not limited to the exactdetails shown and described, for variations obvious to one skilled inthe art will be included within the invention defined by the claims.

1. An isolated DNA molecule comprising a nucleotide sequence of SEQ IDNO:1 or complement thereof.
 2. An isolated DNA molecule capable ofhybridizing to SEQ ID NO:1, or a complement thereof, under hybridizationconditions of 0.015 M NaCl/0.0015 M sodium citrate (SSC) and about 0.1%sodium dodecyl sulfate (SDS) at about 50° C. to about 65° C., whereinthe DNA molecule encodes a polypeptide that is a secretory signalsequence of Pichia methanolica.
 3. The isolated DNA molecule of claim 2wherein the encoded polypeptide has at least 20 contiguous amino acidsof SEQ ID NO:2.
 4. The isolated DNA molecule of claim 2 wherein theencoded polypeptide is SEQ ID NO:2.
 5. An isolated DNA molecule encodinga polypeptide wherein the encoded polypeptide comprises an amino acidsequence having at least 95 percent sequence identity to SEQ ID NO:2,wherein the encoded polypeptide is a secretory signal sequence of Pichiamethanolica.
 6. The isolated DNA molecule of claim 5 wherein the encodedpolypeptide comprises SEQ ID NO:2.
 7. The isolated DNA molecule of claim5 wherein the encoded polypeptide is SEQ ID NO:2.
 8. A DNA constructcomprising the following operably linked elements: a first DNA segmentcomprising a transcription promoter of Pichia methanolica; a second DNAsegment comprising a nucleotide sequence encoding a polypeptide of SEQID NO:2; a third DNA segment encoding a heterologous protein ofinterest; and a fourth DNA segment comprising a transcription terminatorof Pichia methanolica.
 9. The DNA construct of claim 8 wherein the firstDNA segment is a Pichia methanolica transcription promoter selected fromthe group consisting of glyceraldehyde-3-phosphate dehydrogenase 1(GAP1), glyceraldehyde-3-phosphate dehydrogenase 2 (GAP2), alcoholutilization gene 1 (AUG1), and alcohol utilization gene 2 (AUG2). 10.The DNA construct of claim 8 wherein the second DNA segment comprisesSEQ ID NO:1.
 11. The DNA construct of claim 8 wherein the second DNAsegment is SEQ ID NO:1.
 12. The DNA construct of claim 8 wherein thefourth DNA segment comprises a Pichia methanolica transcriptionterminator selected from the group of genes consisting of GAP1, GAP2,AUG1, and AUG2.
 13. The DNA construct of claim 8 further comprising aselectable marker.
 14. The DNA construct of claim 13 wherein theselectable marker is an ADE2 gene.
 15. The DNA construct of claim 8further comprising a Pichia methanolica origin of replication.
 16. TheDNA construct of claim 8 further comprising a fifth operably linked DNAsegment wherein the fifth DNA segment comprises an affinity tag, atherapeutic agent or a detectable label.
 17. The DNA construct of claim8 further comprising a fifth operably linked DNA segment wherein thefifth DNA segment comprises an immunoglobulin moiety comprising at leastone constant region.
 18. The DNA construct of claim 17 wherein the fifthDNA segment is a human immunoglobulin Fc fragment.
 19. A Pichiamethanolica cell containing the DNA construct of claim
 8. 20. The Pichiamethanolica cell of claim 19 wherein the DNA construct is genomicallyintegrated.
 21. The Pichia methanolica cell of claim 20 wherein the DNAconstruct is genomically integrated in multiple copies.
 22. The Pichiamethanolica cell of claim 19 wherein the cell is functionally deficientin vacuolar proteinase A.
 23. The Pichia methanolica cell of claim 19wherein the cell is functionally deficient in vacuolar proteinase B. 24.The Pichia methanolica cell of claim 19 wherein the cell is functionallydeficient in vacuolar protease A and vacuolar protease B.
 25. The Pichiamethanolica cell of claim 19 wherein the cell comprises a functionallydeficient AUG1 gene.
 26. The Pichia methanolica cell of claim 19 whereinthe cell comprises a functionally deficient AUG2 gene.
 27. The Pichiamethanolica cell of claim 19 wherein the cell comprises functionallydeficient AUG1 and AUG2 genes.
 28. A method of producing a protein ofinterest comprising: culturing the cell of claim 19 wherein the thirdDNA segment is expressed and the protein of interest is produced; andrecovering the protein of interest.